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WO2016090585A1 - 测序数据处理装置和方法 - Google Patents

测序数据处理装置和方法 Download PDF

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Publication number
WO2016090585A1
WO2016090585A1 PCT/CN2014/093516 CN2014093516W WO2016090585A1 WO 2016090585 A1 WO2016090585 A1 WO 2016090585A1 CN 2014093516 W CN2014093516 W CN 2014093516W WO 2016090585 A1 WO2016090585 A1 WO 2016090585A1
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Prior art keywords
sequence
read
sequencing
result
pair
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English (en)
French (fr)
Inventor
刘兴民
刘敬一
刘耿
赵鑫
杨明
侯勇
吴逵
李波
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BGI Shenzhen Co Ltd
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BGI Shenzhen Co Ltd
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Priority to PCT/CN2014/093516 priority Critical patent/WO2016090585A1/zh
Priority to CN201480082792.XA priority patent/CN107077538B/zh
Publication of WO2016090585A1 publication Critical patent/WO2016090585A1/zh
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B30/00—ICT specially adapted for sequence analysis involving nucleotides or amino acids

Definitions

  • the present invention relates to the field of biological information. Specifically, the present invention relates to a sequencing data processing apparatus and method, and more particularly, to a sequencing data processing apparatus, a sequencing data processing system, and a processing method for sequencing data.
  • cfDNA (cell-free DNA), which is present in serum, plasma or other body fluids, is an effective biomarker that can be applied to a variety of mutation detection, such as cancer, fetal chromosomal variation and other genetic mutations. Due to the lack of high sensitivity and accuracy of quantitative analysis techniques, previous studies have focused on a number of known disease-related genes, such as the pigmentoma-GNAQ gene (Metz, Stephan HD, et al. Ultradeep sequencing detection GNAQ and GNA11mutations). In cell ⁇ free DNA from plasma of patients with uveal melanoma. Cancer medicine 2.2 (2013): 208-215.), 21 Trisomy 21 (Liao, Gary JW, et al. "Noninvasive prenatal diagnosis of fetal trisomy 21by Allelic ratio analysis using targeted massively parallel sequencing of maternal plasma DNA. "PLoS One 7.5 (2012): e38154.) and the like.
  • MPS Massively Parallel Sequencing
  • SNP Single Nucleotide Polymorphism
  • SNP may cause a variety of human diseases, such as cancer, infectious diseases (AIDS, leprosy, hepatitis, etc.), autoimmune diseases, neuropsychiatric diseases, sickle cell anemia, beta thalassemia and cystic fibrosis.
  • SNPs that have no effect on phenotype, due to their stable inheritance in generations, are important in genome-wide association studies (GWAS) [Thomas, Philippe E., et al. "Challenges in the association of human single nucleotide polymorphism mentions with unique database identifiers. "BMC bioinformatics 12. Suppl 4 (2011): S4.]. Therefore, SNPs are called third-generation genetic markers and have been extensively studied.
  • GWAS genome-wide association studies
  • the present invention is directed to solving at least some of the above technical problems or at least providing a commercial choice.
  • the present invention provides a sequencing data processing apparatus, the apparatus comprising: a data receiving unit, configured to receive the sequencing data, the sequencing data comprising a plurality of pairs of read pairs, each pair of reads Composed of two reads, each from two positions of a chromosome segment, two reads of each pair of read pairs are from the positive and negative strands of the chromosome segment, or each pair of reads Both reads are from the positive strand of the chromosome fragment or the negative strand of the chromosome, each read contains a gap, and the two reads of a pair of read pairs are defined as the left arm and the right arm, respectively; And executing the data processing program, the executing the data processing program includes implementing the comparison of the sequencing data with a reference sequence, obtaining a comparison result, and eliminating a gap of each of the comparison results.
  • the pair of reads from two positions of a chromosome fragment, respectively, can be obtained by sequencing a constructed library by constructing a pair-end library or a mate-pair library.
  • multiple pairs of read pairs are obtained using the library construction method of Complete Genomics (CG) and its sequencing platform. The distance between a pair of read pairs is determined by the length of the read and the enzyme.
  • CG Complete Genomics
  • the distance between the recognition site and the cleavage site is controlled.
  • the CG platform was constructed by enzymatic cleavage to construct a multi-linker paired-end library, and the constructed circular library was sequenced by a unique combinatorial probe-ligation sequencing (cPAL) technique.
  • cPAL combinatorial probe-ligation sequencing
  • Two segments of a linker are used to construct a paired-end library, since each enzyme has a preferred cutting distance, and in actual digestion, it is often one more position or one less than the preferred distance, which makes the reading often With a gap, the gap is often +1 or -1, and / or, if the same enzyme is used for multiple digestions during the construction of the library, the position of the enzyme digestion is easy to change, and the position of the enzyme digestion will change.
  • the obtained reads are nicked, for example, when constructing a multi-ligand circular library, the Alu enzyme is used for two digestions to join different portions of the plurality of linkers, and when the bases adjacent to the linkers are read, a band of +3 is generated. A reading of the gap of /-3.
  • the size of the gap in the present invention may also be zero.
  • the 2-AD sequencing output has a total length of 60 bp, which can be divided into two pairs of mate-paired reads, and each pair of reads is centered.
  • the reads have a small gap at 10 bp, an invalid sequencing site N at the 20 bp position, and the distance between the two reads of a pair of reads is generally less than 2000 bp. From a plurality of reads in a multi-joint library, one read can form a pair of read pairs with any other read.
  • positive strand and negative strand are complementary two strands constituting a chromosome fragment, and are opposite.
  • a strand is said to be a positive strand, and its complementary strand may be referred to as a negative strand, in an embodiment of the present invention.
  • a chain that matches a reference sequence is referred to as a positive chain, and another chain is referred to as a negative chain.
  • the alignment can be performed using known comparison software, such as SOAP, BWA, etc., can also be performed using the comparison software TeraMap of the CG platform.
  • the alignment is performed using TeraMap, and the resulting alignment result is in the format TeraMap.
  • the gap of each read in the elimination comparison result means that the negative gap is removed from the read with the negative gap, that is, the overlapping base is removed, and the positive gap is removed.
  • the read segment replaces the size of the positive gap by N, N is A, T, C or G.
  • N is A, T, C or G.
  • the read can be divided into two parts based on the gap, the end of the two parts There are 2 nt overlaps.
  • the two parts of the read are ATCGCTTAAG and AGTACGATTC respectively, and the negative gaps are overlapped, and the corresponding read is ATCGCTTAAGTACGATTC.
  • the aligning in the method of one aspect of the invention comprises: comparing the left and right arms of each pair of read pairs to the reference sequence, respectively, to obtain a level one left alignment The result is compared with the first-order right-aligned result; one of the first-order left-aligned result and the first-order right-aligned result is used as a reference, and the other is compared, and the second-order left-aligned result and the second are obtained.
  • the read comparison result can be obtained.
  • the first alignment is globally aligned with the reference sequence
  • the left arm/right arm alignment result is The second alignment of the baseline for the right arm/left arm alignment results is a local alignment, such that alignments from the second-order left alignment result and the second-order right alignment result, respectively, can be performed on the same chromosome. The distance between the two reads that match the expected pair is paired into a pair of read pairs, and the read contrast is obtained.
  • the comparing comprises: setting the size of the notch to compare each left arm or each right arm with the reference sequence multiple times to obtain an optimal ratio For the result.
  • the gaps of each of the left arms or each of the right arms are set to -3 nt, -2 nt, -1 nt, 0 nt, 1 nt, 2 nt, 3 nt, 4 nt, 5 nt, 6 nt, and 7 nt, respectively.
  • a read segment respectively comparing the corresponding plurality of read segments with the reference sequence, and using the optimal aligned sequence as the left arm/right arm, where the comparison result may be based on the utilized Compare the software to the default evaluation of the results.
  • executing the data processing program further includes implementing, before the gap of each of the comparison results in the comparison result, extracting a unique comparison result in the comparison result to replace The alignment result, the unique alignment result comprising a plurality of read pairs uniquely aligned with the reference sequence, and each of the reads contrasts to the same chromosome to the reference sequence, each of the The distance between the two reads of the pair of reads corresponds to the expected distance between the two locations of the chromosome segment from which it came.
  • executing the data processing program further comprises implementing correcting a positive strand of the same chromosome that contrasts each pair of the unique alignment results to the reference sequence. For example, for a pair of reads that respectively align the positive and negative strands of the previous chromosome, the reads of the aligned negative strands become their complementary strands, thus replacing the reads with their reverse complementary strands. Said correction.
  • executing the data processing program further comprises implementing data format conversion, the number
  • the format conversion includes converting the format of the alignment result or the unique alignment result.
  • the format of the general comparison result is required to be SAM or BAM, so as to facilitate subsequent analysis of the data based on the comparison result or the comparison result
  • SAM or BAM is a common binary format
  • BAM is a SAM. Compressed format. Due to the use of different comparison software, the format of the output comparison result or the unique comparison result may not be applicable to existing subsequent data processing or analysis software programs, such as the comparison result of the aforementioned TeraMap format, and the output data format thereof. It does not meet the requirements of the input data format of most existing mutation detection software SOAPsnp, GATK or SOAPindel, and converts the data format to obtain the general comparison result with the common data format, which is convenient for further analysis and processing of the data.
  • a sequencing data processing system comprising a host and a display, the system further comprising a sequencing data processing device in accordance with one or any embodiment of the present invention.
  • a method for processing a sequencing data comprising the steps of: acquiring sequencing data, the sequencing data comprising a plurality of pairs of read segments, each pair of read segments consisting of two read segments, respectively Two positions from one chromosome segment, two reads from each pair of reads are from the positive and negative strands of the chromosome segment, or two reads from each pair of read pairs are from the chromosome a positive strand of a fragment or a negative strand of the chromosome fragment, each read comprising a gap, defining two reads of a pair of read pairs as a left arm and a right arm, respectively; comparing the sequencing data to a reference sequence And obtaining a comparison result, the comparison result comprising a comparison result of the plurality of the pair of readings, and/or, the comparison result comprising a comparison result of the plurality of the left arms and a plurality of the The result of the alignment of the right arm; the gap of each of the
  • the pair of reads from two positions of a chromosome fragment can be constructed by constructing a pair-end library or a mate-pair library.
  • sequencing in one embodiment of the present invention, multiple pairs of read pairs are obtained by using the library construction method of Complete Genomics (CG) and its sequencing platform, and the distance between a pair of read pairs is read by The length and the distance between the recognition site of the enzyme and the cleavage site are controlled.
  • CG Complete Genomics
  • the CG platform was constructed by enzymatic cleavage to construct a multi-linker paired-end library, and the constructed circular library was sequenced by a unique combinatorial probe-ligation sequencing (cPAL) technique.
  • the bases on both sides of the linker were read because they were ligated by restriction enzyme digestion.
  • Two segments of a linker are used to construct a paired-end library, since each enzyme has a preferred cutting distance, and in actual digestion, it is often one more position or one less than the preferred distance, which makes the reading often With a gap, the gap is often +1 or -1, and / or, if the same enzyme is used for multiple digestions during the construction of the library, the position of the enzyme digestion is easy to change, and the position of the enzyme digestion will change.
  • the obtained reads are nicked, for example, when constructing a multi-ligand circular library, the Alu enzyme is used for two digestions to join different portions of the plurality of linkers, and when the bases adjacent to the linkers are read, a band of +3 is generated. A reading of the gap of /-3.
  • the size of the gap in the present invention may also be zero.
  • Multiple reads from a multi-ligand library, one read can and either Its read segments form a pair of read pairs.
  • the term "positive strand” and "negative strand” as used herein are complementary two strands constituting a chromosome fragment, and are opposite.
  • a strand is said to be a positive strand, and its complementary strand may be referred to as a negative strand, in an embodiment of the present invention.
  • a chain that matches a reference sequence is referred to as a positive chain, and another chain is referred to as a negative chain.
  • the alignment can be performed using known comparison software, such as SOAP, BWA, etc., or can be performed using the comparison software TeraMap of the CG platform.
  • the alignment is performed using TeraMap, and the resulting alignment result is in the format TeraMap.
  • the gap of each read in the elimination comparison result means that the negative gap is removed from the read with the negative gap, that is, the overlapping base is removed, and the positive gap is removed.
  • the read segment replaces the size of the positive gap by N, N is A, T, C or G.
  • N is A, T, C or G.
  • the read can be divided into two parts based on the gap, the end of the two parts There are 2 nt overlaps.
  • the two parts of the read are ATCGCTTAAG and AGTACGATTC respectively, and the negative gaps are overlapped, and the corresponding read is ATCGCTTAAGTACGATTC.
  • obtaining the sequencing data comprises constructing a sequencing library to obtain a sequencing library, the sequencing library being a single-stranded circular DNA library, the sequencing library being composed of a strand of the chromosome fragment and at least one The predetermined DNA sequence is constructed.
  • the single-stranded circular library can be constructed by a known library construction method, for example, a single-linker circular double-stranded library is obtained by reference to the construction of a paired-end library of SOLiD of Life Technologies, and then the double-stranded single-stranded circular library is obtained.
  • the single-stranded circular library is constructed using the CG library construction technique, and the library construction can be referred to US7897344 to obtain a multi-linker single-stranded circular library.
  • each pair of reads is from both ends of the chromosome segment.
  • two parts of a linker are respectively ligated to both ends of a chromosome fragment, single-stranded and single-stranded to obtain a 1-ligand single-stranded circular library, and the 1-linker single-stranded
  • the circular library consists of one strand of the chromosome fragment and a predetermined DNA sequence connecting the two ends of the strand, the rolling circle is amplified to form DNA nanospheres (DNB), and the CG platform is sequenced using its unique high density.
  • DNA nanochip technology the principle is to embed DNA nanospheres on the chip, read sequence with non-continuous, non-linked joint probe anchor continuous (cPAL) technology, for DNB embedded into the chip and cPAL technology can refer to US8278039B2 and US8518640B2 respectively .
  • the predetermined DNA sequence is a known sequence and is a link of the aforementioned linker or linker.
  • the improved CG building method constructs a 1-ligand circular single-strand library comprising the steps of: (1) extracting a nucleic acid to be tested; (2) phosphorylating the nucleic acid at the terminal to obtain a terminal phosphorylated product; and (3) end-repairing Said terminal phosphorylation product, obtaining a terminal repair product; (4) linking the first sequence and the second sequence to both ends of the terminal repair product to obtain a first ligation product; (5) using the third sequence for the ligation The product is subjected to nick translation and amplification to obtain an amplification product, the third sequence being a pair of primer pairs, at least one primer of the primer pair carrying a biotin label; (6) using the biotin label to Amplification products are subjected to single-strand separation to obtain a single-stranded product; (7) cyclizing the single-stranded product with a fourth sequence to obtain the sequencing library; wherein the fourth sequence is capable of ligating one end of the first sequence And at one end of the second sequence, the other
  • Said fourth sequence is capable of connecting said first sequence and said second sequence Into a linker, the nick translation is a nick caused by the elimination of the dideoxynucleotide attached to the first end of the end repair product and/or the other end of the second sequence, using at least one primer
  • the biotin label carries at least one strand of the amplified product with a biotin label, making it easier to subsequently isolate the single stranded product based on the biotin label.
  • the improved CG library construction method constructs a 1-ligand circular single-strand library comprising the steps of: (1) extracting a nucleic acid to be tested; (2) repairing the nucleic acid at the end to obtain a terminal repair product.
  • terminal phosphorylating the terminal repair product to obtain a terminal phosphorylation product (3) terminal phosphorylating the terminal repair product to obtain a terminal phosphorylation product; (4) linking the first sequence and the second sequence to both ends of the terminal phosphorylation product to obtain a first ligation product; Performing nick translation and amplification of the ligation product using a third sequence to obtain an amplification product, the third sequence being a pair of primer pairs, at least one primer of the primer pair carrying a biotin label; (6) Single-stranding the amplification product by the biotin label to obtain a single-stranded product; (7) cyclizing the single-stranded product with a fourth sequence to obtain the sequencing library; wherein the fourth sequence One end of the first sequence and one end of the second sequence can be ligated, and the other end of the first sequence and/or the second sequence is a dideoxynucleotide.
  • End repair is to obtain a blunt-ended nucleic acid fragment that enables attachment of other nucleotides or sequences.
  • Terminal phosphorylation is to reduce the interconnection of sample nucleic acid fragments, so that samples with low nucleic acid content can also be constructed in a library and meet the requirements of the library.
  • Single-linker circular single-strand library As shown in Figure 1, the constructed single-linker circular single-strand library (1-AD) was sequenced on the machine, and the 1-AD sequencing output read pair had a total length of about 30 bp, one read. 12 bp, 19 bp in one read, the median distance of the genome between the two reads in a read is about 140 bp.
  • the single joint has a small amount of storage, which is suitable for the case of less cfDNA content, and has the advantages of short construction time and low construction cost.
  • the alignment in the method of the invention comprises: comparing the left and right arms of each pair of read pairs to the reference sequence, respectively, to obtain a level 1 left alignment result and The first-order right-aligned result is compared with one of the first-order left-aligned result and the first-order right-aligned result, and the other is compared, and the second-order left-aligned result and the second-level right are obtained. Aligning the results, obtaining a comparison result of the plurality of the pair of readings based on the result of the second-order left alignment and the result of the second-order right alignment, or obtaining a comparison result of the plurality of the left arms and The alignment of the right arms.
  • the read comparison result can be obtained.
  • the first alignment is globally aligned with the reference sequence
  • the left arm/right arm alignment result is The second alignment of the baseline for the right arm/left arm alignment results is a local alignment, such that alignments from the second-order left alignment result and the second-order right alignment result, respectively, can be performed on the same chromosome.
  • the distance between the two reads that match the expected pair is paired into a pair of read pairs, and the read contrast is obtained.
  • the aligning includes arranging the gaps such that each left or each right arm is compared with the reference sequence multiple times to obtain an optimal alignment result.
  • the gaps of each of the left arms or each of the right arms are set to -3 nt, -2 nt, -1 nt, 0 nt, 1 nt, 2 nt, 3 nt, 4 nt, 5 nt, 6 nt, and 7 nt, respectively.
  • a read segment respectively comparing the corresponding plurality of read segments with the reference sequence, and using the optimal aligned sequence as the left arm/right arm, where the comparison result may be based on the utilized Comparison software comparison result The default judgement.
  • executing the data processing program further includes implementing, before the gap of each of the comparison results in the comparison result, extracting a unique comparison result in the comparison result to replace The alignment result, the unique alignment result comprising a plurality of read pairs uniquely aligned with the reference sequence, and each of the reads contrasts to the same chromosome to the reference sequence, each of the The distance between the two reads of the pair of reads corresponds to the expected distance between the two locations of the chromosome segment from which it came.
  • executing the data processing program further comprises implementing correcting a positive strand of the same chromosome that contrasts each pair of the unique alignment results to the reference sequence. For example, for a pair of reads that respectively align the positive and negative strands of the previous chromosome, the reads of the aligned negative strands become their complementary strands, thus replacing the reads with their reverse complementary strands. Said correction.
  • executing the data processing program further comprises implementing a data format conversion, the data format conversion comprising converting the alignment result or the format of the unique alignment result.
  • the format of the general comparison result is required to be SAM or BAM, so as to facilitate subsequent analysis of the data based on the comparison result or the comparison result
  • SAM or BAM is a common binary format
  • BAM is a SAM. Compressed format. Due to the use of different comparison software, the format of the output comparison result or the unique comparison result may not be applicable to existing subsequent data processing or analysis software programs, such as the comparison result of the aforementioned TeraMap format, and the output data format thereof. It does not meet the requirements of the input data format of most existing mutation detection software SOAPsnp, GATK or SOAPindel, and converts the data format to obtain the general comparison result with the common data format, which is convenient for further analysis and processing of the data.
  • a computer readable storage medium for storing a program for execution by a computer, the execution of the program comprising performing an aspect of the aforementioned invention or any one of its embodiments. Sequencing data processing method.
  • the foregoing description of the advantages and technical features of the sequencing data processing method of the present invention is also applicable to the computer readable storage medium, and details are not described herein again.
  • the storage medium may include: a read only memory, a random access memory, a magnetic disk or an optical disk, and the like.
  • the invention provides a method for detecting a single nucleotide polymorphism (SNP), the method comprising: A. obtaining a nucleic acid of a sample to be tested; B. performing at least a portion of the nucleic acid Sequencing, obtaining sequencing data; C. processing the sequencing data to obtain a universal alignment result; D. detecting a SNP based on the universal comparison result; wherein, step C is utilized in one aspect of the invention or any specific The sequencing data processing apparatus and/or method in the embodiments are performed.
  • SNP single nucleotide polymorphism
  • the step B comprises performing a sequencing library construction on at least a portion of the nucleic acid to obtain a sequencing library, the sequencing library being a single-stranded circular DNA library, the single-stranded circular DNA library
  • the construct comprises: terminal phosphorylating the nucleic acid to obtain a terminal phosphorylation product; terminally repairing the terminal phosphorylation product to obtain an end repair a multiplex product; a first sequence and a second sequence are ligated to both ends of the terminal repair product to obtain a first ligation product; and the ligation product is subjected to nick translation and amplification using a third sequence to obtain an amplification product,
  • the third sequence is a pair of primer pairs, at least one primer of the primer pair carrying a biotin label; the amplification product is subjected to single-strand separation using the biotin label to obtain a single-stranded product; a single-stranded product, wherein the sequencing library is obtained; wherein the fourth sequence is capable of joining one end
  • end repair is performed followed by terminal phosphorylation.
  • End repair is to obtain a blunt-ended nucleic acid fragment that enables attachment of other nucleotides or sequences.
  • Terminal phosphorylation is to reduce the interconnection of sample nucleic acid fragments, so that samples with low nucleic acid content can also be constructed in a library and meet the requirements of the library.
  • Said fourth sequence is capable of joining the first sequence and the second sequence to form one of said linkers, and the nick translation is for eliminating the dideoxy core attached to the first end of the end repair product and/or the other end of the second sequence
  • a nick caused by a glycoside with at least one primer carrying a biotin label to carry at least one strand of the amplified product with a biotin label, so that subsequent separation of the single-stranded product based on the biotin label is easily obtained.
  • the single-linker circular single-stranded library is shown in Figure 1.
  • the single-linker has a small amount of storage, which is suitable for the case of less cfDNA content. In addition, it has the advantages of short construction time and low cost of construction.
  • sequencing of the constructed library is performed using a combinatorial probe anchoring ligation sequencing technique, for example, using a CG sequencing platform, which avoids the accumulation of errors in sequencing, and its accuracy is more synthetic.
  • the method and the ligation method have high sequencing, up to 99.999%, and the sequencing data is obtained by single-link sequencing of the CG platform, and the cost is lower and the speed is faster.
  • the detection of SNPs based on universal alignment results can utilize currently known SNP detection methods and/or software, such as using SOAP2, samtools, GATK, and the like.
  • the open source software samtools is used to preprocess the common alignment results (bam files), including sorting, removing duplicate reads generated by the database PCR, and then using samtools mpileup and/or GATK open source software. Get the SNP results and convert them to vvc format using the open source software bcftools. Compared to other tools, Samtools is simple to operate, and the output format is common. Multi-threading can be used to improve efficiency during big data processing.
  • the present invention provides a SNP detecting apparatus for performing all or part of the steps of the SNP detecting method of one aspect of the present invention, the apparatus comprising: a nucleic acid acquiring apparatus for acquiring a test a nucleic acid of the sample; a sequencing device for sequencing at least a portion of the nucleic acid from the nucleic acid acquisition unit to obtain sequencing data, the sequencing data comprising a plurality of pairs of read pairs, each pair of read pairs consisting of two reads , respectively, from two positions of a chromosome segment, two reads in each pair of read pairs are from the positive and negative strands of the chromosome segment, or two reads from each pair of read pairs are from a positive strand of a chromosome fragment or a negative strand of the chromosome, each read containing a gap, defining two reads of a pair of read pairs as a left arm and a right arm, respectively; a data processing device for pairing The sequencing data of
  • the data processing apparatus, system and/or method of the invention comprises the following steps: developing the TeraMap2Sam conversion software, accurately processing the gap in the sequencing data, and converting the comparison result of the CG platform TeraMap into a general SAM format, so that the subsequent use of the Samtools can be directly used. , GATK and many other excellent open source software for mutation detection, making the selection of subsequent analysis more extensive.
  • the SNP detection method and/or device of the present invention including Sumtools software for SNP analysis, is simple, universal, fast, and highly reliable.
  • FIG. 1 is a schematic view showing the structure of a single-linker circular single-stranded library in one embodiment of the present invention
  • FIG. 2 is a schematic structural diagram of a sequencing data processing apparatus in an embodiment of the present invention.
  • FIG. 3 is a schematic structural diagram of a sequencing data processing system in an embodiment of the present invention.
  • FIG. 4 is a flow chart of a method for processing sequencing data in an embodiment of the present invention.
  • Figure 5 is a flow chart showing a method of processing sequencing data in an embodiment of the present invention.
  • FIG. 6 is a flow chart of a SNP detecting method in one embodiment of the present invention.
  • FIG. 7 is a schematic structural diagram of a SNP detecting apparatus in an embodiment of the present invention.
  • Figure 8 is a flow diagram showing the construction and sequencing of a single linker library in one embodiment of the present invention.
  • Figure 9 is a flow chart of SNP detection in an embodiment of the present invention.
  • Figure 10 is a flow chart of the algorithm of the Teramap2Sam software in one embodiment of the present invention.
  • the sequencing data processing apparatus 100 includes a data receiving unit 10, a processor 20, and a storage unit 30, a processor 20 and a data receiving unit 10, and The storage unit 30 is connected, and the storage unit 30 is connected to the data processing unit 10.
  • the data receiving unit 10 is configured to receive sequencing data, where the sequencing data includes multiple pairs of read pairs, each pair of read segments consists of two read segments, respectively, from two positions of a chromosome segment, each pair of read long pairs
  • the two reads are from the positive and negative strands of the chromosome fragment, respectively, or both reads in each pair of reads are from the positive strand of the chromosome fragment or the negative strand of the chromosome, each read
  • the segments all contain gaps, and the two reads of a pair of read pairs are defined as the left and right arms, respectively.
  • the pair of reads from two positions of a chromosome fragment, respectively can be obtained by sequencing a constructed library by constructing a pair-end library or a mate-pair library.
  • multiple pairs of read pairs are obtained using the library construction method of Complete Genomics (CG) and its sequencing platform.
  • the distance between a pair of read pairs is determined by the length of the read and the enzyme.
  • the distance between the recognition site and the cleavage site is controlled.
  • the CG platform was constructed by enzymatic cleavage to construct a multi-linker paired-end library, and the constructed circular library was sequenced by a unique combinatorial probe-ligation sequencing (cPAL) technique.
  • cPAL combinatorial probe-ligation sequencing
  • Two segments of a linker are used to construct a paired-end library, since each enzyme has a preferred cutting distance, and in actual digestion, it is often one more position or one less than the preferred distance, which makes the reading often With a gap, the gap is often +1 or -1, and / or, if the same enzyme is used for multiple digestions during the construction of the library, the position of the enzyme digestion is easy to change, and the position of the enzyme digestion will change.
  • the obtained reads are nicked, for example, when constructing a multi-ligand circular library, the Alu enzyme is used for two digestions to join different portions of the plurality of linkers, and when the bases adjacent to the linkers are read, a band of +3 is generated. A reading of the gap of /-3.
  • the size of the gap in the present invention may also be zero.
  • the 2-AD sequencing output has a total length of 60 bp, which can be divided into two pairs of mate-paired reads, and each pair of reads is centered.
  • the reads have a small gap at 10 bp, an invalid sequencing site N at the 20 bp position, and the distance between the two reads of a pair of reads is generally less than 2000 bp. From a plurality of reads in a multi-joint library, one read can form a pair of read pairs with any other read.
  • positive strand and negative strand are complementary two strands constituting a chromosome fragment, and are opposite.
  • a strand is said to be a positive strand, and its complementary strand may be referred to as a negative strand, in an embodiment of the present invention.
  • a chain that matches a reference sequence is referred to as a positive chain, and another chain is referred to as a negative chain.
  • the processor 20 is configured to execute a data processing program, and the executing the data processing program comprises: comparing the sequencing data with a reference sequence, obtaining a comparison result, and eliminating each read in the comparison result a gap, obtaining a universal alignment result, the alignment result comprising a plurality of alignment results of the pair of reads, and/or, the comparison result comprising a plurality of Alignment results of the left arms and alignment results of a plurality of the right arms.
  • the comparison can be performed by using known comparison software, such as SOAP, BWA, etc., or by using the comparison software TeraMap of the CG platform.
  • the alignment is performed using TeraMap, and the resulting alignment result is in the format TeraMap.
  • the gap of each read in the elimination comparison result means that the negative gap is removed from the read with the negative gap, that is, the overlapping base is removed, and the positive gap is removed.
  • the read segment replaces the size of the positive gap by N, N is A, T, C or G, and the read with the gap 0 is not processed.
  • N is A, T, C or G
  • the read with the gap 0 is not processed.
  • the read based on the gap The segment can be divided into two parts, and the ends of the two parts have 2nt overlap.
  • the two parts of the read segment are ATCGCTTAAG and AGTACGATTC respectively, and the negative gap, that is, the overlapping AG, is eliminated, and the corresponding read segment is obtained as ATCGCTTAAGTACGATTC.
  • the storage unit 30 is for storing data, and the above-described data processing program is stored in the storage unit 30, and intermediate data or results of the processing of the sequencing data from the data receiving unit 10 and the processor 20 are also stored.
  • FIG. 3 is a block diagram showing the structure of a system in an embodiment of the sequencing data processing system of the present invention.
  • the sequencing data processing system 1000 includes a sequencing data processing device 100, a host 200, and a display device 300.
  • the host 200 can be an audio/video/signal source device, such as a computer host, mainframe, etc., for transmitting display data required by the display device 300.
  • the host 200 includes at least one interface electrically connected to the sequencing data processing device 100.
  • the sequencing data processing device 100 receives the sequencing data output from the host 200, processes the sequenced data, and then outputs the processed data or results to the display device. 300.
  • the sequencing data processing method comprises the steps of: S1 acquiring sequencing data, the sequencing data comprising a plurality of pairs of read segments, each pair of read segments consisting of two read segments, respectively, from two positions of a chromosome segment, each pair of reads The two reads of the pair are from the positive and negative strands of the chromosome fragment, respectively, or both reads of each pair of read lengths are from the positive strand of the chromosome fragment or the negative strand of the chromosome fragment, Each read segment includes a gap, and two reads of a pair of read pairs are respectively defined as a left arm and a right arm; S2 compares the sequencing data with a reference sequence to obtain a comparison result, and the comparison result Include a comparison result of a plurality of the pair of read segments, and/or, the comparison result includes a comparison result of the plurality of the left arms and a comparison result of the plurality of the
  • the pair of reads from two positions of a chromosome fragment can be constructed by constructing a pair-end library or a mate-pair library.
  • multiple pairs of read pairs are obtained by using the library construction method of Complete Genomics (CG) and its sequencing platform, and the distance between a pair of read pairs is read by The length and the distance between the recognition site of the enzyme and the cleavage site are controlled.
  • CG platform to construct a multi-linker paired end library by enzymatic digestion
  • the constructed circular library was sequenced by a unique combinatorial probe-ligation sequencing (cPAL) technique, and the bases on both sides of the linker were read, as it was constructed by pairing two ends of a linker to perform paired-end library construction.
  • cPAL combinatorial probe-ligation sequencing
  • each enzyme has a preferred cutting distance, and often there is one more position or one less position than the preferred distance in the actual enzymatic cutting, so that the reading often has a gap, and the gap is often +1. Or -1, and / or, if the same enzyme is used for multiple digestions during the construction of the library, the position of the enzyme digestion is easy to change each time, and the change of the enzyme digestion position will also cause the obtained reads to have a gap, for example, in the construction.
  • the Alu enzyme is digested twice to join different portions of the plurality of adaptors, and when the bases next to these linkers are read, a read with a gap of +3/-3 is generated.
  • the size of the gap in the present invention may also be zero. From a plurality of reads in a multi-joint library, one read can form a pair of read pairs with any other read.
  • the term "positive strand” and "negative strand” as used herein are complementary two strands constituting a chromosome fragment, and are opposite. When a strand is a positive strand, the complementary strand can be said to be a minus strand.
  • a chain that matches a reference sequence is referred to as a positive chain, and another chain is referred to as a negative chain.
  • the comparison can be performed by using known comparison software, such as SOAP, BWA, etc., or by using the comparison software TeraMap of the CG platform.
  • the alignment is performed using TeraMap, and the resulting alignment result is in the format TeraMap.
  • the gap of each read in the elimination comparison result means that the negative gap is removed from the read with the negative gap, that is, the overlapping base is removed, and the positive gap is removed.
  • the read segment replaces the size of the positive gap by N, N is A, T, C or G, and the read with the gap 0 is not processed.
  • the read based on the gap The segment can be divided into two parts, and the ends of the two parts have 2nt overlap.
  • the two parts of the read segment are ATCGCTTAAG and AGTACGATTC respectively, and the negative gap, that is, the overlapping AG, is eliminated, and the corresponding read segment is obtained as ATCGCTTAAGTACGATTC.
  • FIG. 5 is a flow chart showing the data processing of one embodiment of the sequencing data processing method of the present invention.
  • the sequencing data processing method comprises: S10 acquiring sequencing data, the sequencing data comprising a plurality of pairs of read pairs, each pair of read segments consisting of two read segments, respectively, from two positions of one chromosome segment, each pair of read pairs The two reads in the pair are from the positive and negative strands of the chromosome fragment, or the two reads in each pair of read lengths are from the positive strand of the chromosome fragment or the negative strand of the chromosome fragment, each Each of the read segments includes a gap, and two reads of a pair of read pairs are respectively defined as a left arm and a right arm; S20 compares the sequencing data with a reference sequence to obtain a comparison result, and the comparison result includes Aligning results of a plurality of the pair of read segments, and/or, the comparison result includes a comparison result of the plurality of the left arms and a comparison result of the plurality
  • Fig. 6 is a flow chart showing the detection of an embodiment of the SNP detecting method of the present invention.
  • the SNP detection method comprises the steps of: S11 acquiring nucleic acid of a sample to be tested; S12 sequencing the nucleic acid to obtain sequencing data; S13 processing the sequencing data to obtain a general comparison result; S14 is based on the universal comparison As a result, the SNP is detected; wherein S13 is performed using a sequencing data processing device and/or a sequencing data processing method in one aspect of the invention or in any one of the embodiments.
  • the detection of SNPs based on universal alignment results can utilize currently known SNP detection methods and/or software tools, such as using SOAP2, GATK, samtools, and the like.
  • FIG. 7 is a schematic structural diagram of an apparatus of an embodiment of a SNP detecting apparatus of the present invention.
  • the device 2000 includes: a nucleic acid acquiring device 200 for acquiring nucleic acid of a sample to be tested; and a sequencing device 400 for sequencing nucleic acid from the nucleic acid acquiring unit to obtain sequencing data, the sequencing data including multiple pairs of readings Yes, each pair of reads consists of two reads, one from each of the two chromosome segments, and two of each pair of read pairs are from the positive and negative strands of the chromosome segment, respectively.
  • Both reads of the pair of read lengths are from the positive strand of the chromosome fragment or the negative strand of the chromosome, each read contains a gap, and the two reads of a pair of read pairs are respectively defined as left An arm and a right arm; a data processing device 600 for processing sequencing data from the sequencing device to obtain a general alignment result; and a detecting device 800 for based on a general comparison result from the data processing device 600 Detecting a SNP; wherein the data processing apparatus 600 includes: a data receiving unit 610, configured to receive sequencing data from the sequencing apparatus, and a processor 630, configured to execute a data processing program, execute the The data processing program includes implementing the alignment of the sequencing data with a reference sequence, obtaining a comparison result, and eliminating a gap of each of the comparison results, obtaining a universal alignment result, the comparison result Include a comparison result of a plurality of the pair of read segments, and/or, the result of the comparison includes a comparison result of the plurality of the left arms and
  • peripheral blood plasma of lung cancer patients was taken as the test object.
  • the samples were from Southwest Hospital and tested as follows:
  • the above reaction product was purified by 60 ul of Ampure XP beads and eluted with 22 ul of Elution buffer.
  • the above reaction product was purified by 40 ul of Ampure XPbeads and eluted with 22 ul of Elution buffer.
  • the two strands of the first sequence are: TTGGCCTCCGACT/3-ddT/(SEQ ID NO: 1),
  • the two strands of the second sequence are: /5Phos/GTCTCCAGTCGAAGCCCGACG/3ddC/(SEQ ID NO: 3), GCTTCGACTGGAGA/3ddC/(SEQ ID NO: 4).
  • the upstream primer in the third sequence is/5-bio/TCCTAAGACCGCTTGGCCTCCGACT (SEQ ID NO: 5),
  • the intermediate "x" is a variable tag sequence region, which can be replaced by N, N is A, T, C or G, when no other sample libraries are mixed together, only A sample library is on the machine, no tag sequence is required, ie the third sequence can be
  • the target nucleic acid (ctDNA) content in the mixed nucleic acid is low. If a plurality of such sample libraries are mixed and obtained on the machine to obtain mixed data, the split mixed data needs to be corresponding to the respective samples, a part of the data is lost, and the construction is performed.
  • the single-joint circular library reads are relatively short, and accurate sequencing requires deep sequencing to obtain a relatively large amount of assay data, preferably, a single sample library.
  • the above reaction product was purified by 40 ul of Ampure XP beads and eluted with 37.4 ul of Elution buffer.
  • the above reaction product was purified by 50 ul of Ampure XP beads and eluted with 22 ul of Elution buffer.
  • the PCR product was subjected to concentration determination using a Qubit dsDNA HS assay kit.
  • Tween20 0.5% Tween20, 1X BBB/Tween Mix, 1X BWB/Tween Mix, 0.1M NaOH.
  • the 0.5% Tween20 configuration method is the same as the above, and the other three configuration methods are as follows:
  • the product of this step can be stored frozen at -20 °C.
  • ligase reaction mixture is shaken and thoroughly mixed. After centrifugation, 11 ul of the ligase reaction mixture is added to the EP tube to which the primer reaction mixture has been added, shaken for 10 s, and centrifuged instantaneously.
  • the starting amount of the sample used for the preparation of DNB was adjusted to 35.3 ng-53 ng according to the concentration of single-stranded molecular quantitative determination.
  • the corresponding volume sample ( ⁇ 60 ul) was transferred to the Biorad PCR plate, and the total volume was not more than 120 ul using 1XTE. .
  • the final concentration is 5.625-7.5fmol/ul
  • the volume is 120ul
  • the total amount is 35.3ng-53ng
  • the DNB in the 1adapter sequencing needs 120fmol, 7.5foml/ul, 16ul. Therefore, the library needs to be diluted to 7.5 fmol/ul.
  • FIG. 9 is a schematic flowchart of detecting the SNP.
  • the sequencing data processing method and/or SNP detection method of the present invention based on the CG platform sequencing technology, ultra-micro cfDNA enrichment, library establishment, sequencing and data analysis can be performed.
  • the sequencing of the reads is short, and there are resequencing and small gaps at specific locations. It is difficult to directly compare the sequencing results using ordinary comparison software or Detection analysis.
  • the working principle is: First, it will compare the two ends of the read length (LeftArm, RightArm), and TeraMap will try a variety of gaps. The value is used to process the read length to obtain more comparison results; then, the comparison result at each end is taken as a reference, and the other end is locally aligned (for example, 4-AD, the range of the local alignment is 0 to 700bp); if both ends can be well aligned to the same chromosome, and the insert-size meets expectations (eg 4-AD, the distance between the two reads of a read pair is 0-700bp), then only the best alignment result is output Otherwise, multiple comparison results at both ends are output.
  • TeraMap is a comparison software for CG sequencing platform.
  • the Teramap2Sam software is developed according to the method of the present invention, and the gap in the TeraMap comparison result is removed and converted into SAM (sequence alignment/map format).
  • SAM sequence alignment/map format
  • Step 1 Extract the unique alignment results. According to the TeraMap output result matchCount to determine whether the unique alignment, while requiring the length of the insert to meet the requirements and the read alignment of the two ends on a reference sequence.
  • Step 2 Remove the gap.
  • the gap position in the reads is determined according to the gaps field, and the read sequence is corrected.
  • the third step calculate FLAG. According to the comparison direction of the double-ended read, the FLAG parameter in the SAM file is calculated to obtain the comparison.
  • SAM is a more general format for storing comparison information.
  • Each line is a pair of reads. It consists mainly of eleven fields. Later, more fields can be added to contain more information, such as XT:A: U means that this reads is a unique comparison.
  • U means that this reads is a unique comparison.
  • BAM binary compression format
  • CG developed the Assembly Software for its read structure to reassemble the reads, and perform the follow-up work after the assembly is completed.
  • the short readout is short (12 bp).
  • the original CG mutation detection tool is no longer applicable or the detection result is not good.
  • SAM/BAM is a commonly used alignment format for high-throughput sequencing.
  • BAM data can be used to detect SNP mutations.
  • SNP detection can utilize known open source software, such as SOAP2, samtools, GATK, and the like.
  • the open source software samtools is used to preprocess the common alignment results (the SAM format bam file), including sorting, removing duplicate reads generated by the library PCR, and then using the samtools mpileup and/or GATK open source software to obtain the SNP.
  • the open source software bcftools was converted to the vcf format.
  • Samtools is easy to operate, and the output format is common.
  • multi-threading can be used to improve efficiency, and the speed is fast and credible.

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Abstract

一种测序数据处理装置、系统和方法，一种计算机可读存储介质，一种检测SNP的方法及设备，其中，测序数据处理装置包括：数据接收单元（10），用于接收所述测序数据，所述测序数据包括多对读段对，每对读段对由两个读段组成，分别来自一条染色体片段的两个位置，每个读段都包含缺口；处理器（20），用于执行数据处理程序，执行所述数据处理程序包括实现将所述测序数据与参考序列作比对，获得比对结果，以及消除所述比对结果中的每个读段的缺口，获得通用比对结果；以及，至少一个存储单元（30），用于存储数据，其中包括所述数据处理程序。

Description

测序数据处理装置和方法 技术领域
本发明涉及生物信息领域，具体的，本发明涉及测序数据处理装置和方法，更具体地，本发明涉及一种测序数据处理装置、一种测序数据处理系统、一种测序数据的处理方法、一种计算机可读存储介质、一种检测SNP的方法以及一种SNP检测设备。
背景技术
存在于血清、血浆或其他体液中的cfDNA(cell-free DNA)，是一种有效的生物标记物，它可以应用于多种突变检测中，比如癌症、胎儿染色体变异等基因突变导致的疾病。由于缺乏高敏感度和准确度的定量分析技术，此前的大量研究关注的都是一些已知的疾病相关基因，比如色素瘤-GNAQ基因(Metz,Claudia HD,et al.Ultradeep sequencing detects GNAQ and GNA11mutations in cell‐free DNA from plasma of patients with uveal melanoma.Cancer medicine 2.2(2013):208-215.)，21三体-21号染色体(Liao,Gary JW,et al."Noninvasive prenatal diagnosis of fetal trisomy 21by allelic ratio analysis using targeted massively parallel sequencing of maternal plasma DNA."PLoS One 7.5(2012):e38154.)等。
新一代测序技术454(Roche)、Solexa(Illumina)和SOLiD(ABI)等的诞生，使得测序通量迅速提升的而测序成本急剧下降，这为cfDNA检测提供了新的思路。目前大规模并行测序(Massively Parallel Sequencing,MPS)是最主流的cfDNA检测技术，它被广泛应用于血浆DNA分子诊断、胎儿染色体异倍体、全基因组核型分析，甚至胎儿全基因组测序中。单核苷酸多态性(Single Nucleotide Polymorphism，SNP)是指在基因组上单个核苷酸的变异(包括置换、颠换、缺失和插入)，形成的遗传标记，其数量很多，多态性丰富。SNP可能会导致多种人类疾病，如癌症、传染性疾病(艾滋病，麻风病，肝炎等)、自身免疫性疾病、神经精神性疾病、镰状细胞贫血、β地中海贫血症及囊性纤维化等[Ingram,V.M."A Specific Chemical Difference Between the Globins of Normal Human and Sickle-Cell 
Figure PCTCN2014093516-appb-000001
Nature 178(1956).]；与SNP相关的疾病将可能成为药物治疗的主要基因靶点[Fareed,Mohd,and Mohammad Afzal."Single nucleotide polymorphism in genome-wide association of human population:A tool for broad spectrum service."Egyptian Journal of Medical Human Genetics 14.2(2013):123-134.]；某些药物的代谢与SNP紧密相关[Yanase,Kae,et al."Functional SNPs of the breast cancer resistance protein‐therapeutic effects and inhibitor development."Cancer letters 234.1(2006):73-80.]；对表型没有影响的SNP,因其数量在世代中的稳定遗传，在全基因组关联研究(GWAS)中有重要意义[Thomas,Philippe E.,et al."Challenges in the association of human single nucleotide polymorphism mentions with  unique database identifiers."BMC bioinformatics 12.Suppl 4(2011):S4.]。因此SNP被称作第三代遗传标记，被广泛的研究。
发明内容
本发明旨在至少在一定程度上解决上述技术问题之一或至少提供一种商业选择。
依据本发明的第一方面，本发明提出了一种测序数据处理装置，该装置包括：数据接收单元，用于接收所述测序数据，所述测序数据包括多对读段对，每对读段对由两个读段组成，分别来自一条染色体片段的两个位置，每对读长对中的两个读段分别来自所述染色体片段的正链和负链，或者每对读段对中的两个读段都来自所述染色体片段的正链或所述染色体的负链，每个读段都包含缺口，将一对读段对的两个读段分别定义为左臂和右臂；处理器，用于执行数据处理程序，执行所述数据处理程序包括实现将所述测序数据与参考序列作比对，获得比对结果，以及消除所述比对结果中的每个读段的缺口，获得通用比对结果，所述比对结果包括多个所述读段对的比对结果，和/或，所述比对结果包括多个所述左臂的比对结果和多个所述右臂的比对结果；以及，至少一个存储单元，用于存储数据，其中包括所述数据处理程序。这里所说的分别来自一条染色体片段的两个位置的读段对，可以通过构建末端文库(pair-end library)或者配对末端文库(mate-pair library)，对所构建的文库进行测序来获得，在本发明的一个实施例中，利用Complete Genomics公司(CG)的文库构建方法及其测序平台，获得多对读段对，一对读段对之间的距离是由读段的长度以及酶的识别位点和切割位点的距离来控制的。CG平台通过酶切构建多接头配对末端文库，利用特有的组合探针连接测序(cPAL)技术对所构建的环状文库进行测序，测读出接头两旁的碱基，因为其是利用酶切连接一个接头的两段来进行配对末端文库构建的，由于每一种酶都有一个首选的切割距离，而在实际酶切时经常比首选距离多一个位置或少一个位置，这样使得读段中经常带有缺口(gap)，缺口常为+1或者-1，和/或，建库时倘若使用同一种酶多次酶切，每次的酶切位置易发生变化，酶切位置的变化也会使获得的读段带有缺口，例如在构建多接头环状文库时，利用Alu酶两次酶切来连接多个接头的不同部分，读测这些接头旁的碱基时，会产生带+3/-3的缺口的读段。在本发明中缺口的大小还可以是0。以CG平台目前的双接头(two adaptors,2-AD)测序文库为例，2-AD测序输出总长为60bp，可分为两对读段对(mate-paired reads)，每对读段对中的读段在10bp的位置都有小的gap，在20bp位置有一个无效测序位点N，一对读段对的两个读段之间的在基因组上的距离一般小于2000bp。来自多接头文库中的多个读段，一个读段可以和任一其它读段组成一对读段对。这里所说的“正链”和“负链”是组成染色体片段的互补的两条链，是相对的，称一条链为正链就可以称其互补链为负链，在本发明的一个实施例中，将与参考序列匹配的链称为正链，将另一条链称为负链。在本发明中，比对可以利用已知比对软件进行，比如SOAP、 BWA等，也可以利用CG平台的比对软件TeraMap进行。在本发明的一个实施例中，比对是利用TeraMap进行的，所得的比对结果的格式为TeraMap。在本发明的一个实施例中，所说的消除比对结果中每个读段的缺口是指，对带负缺口的读段去除掉其负缺口即去除掉重叠的碱基，对带正缺口的读段以N替代正缺口的大小，N为A、T、C或G，例如，对带负缺口比如为-2nt的读段，基于缺口处该读段可分成两部分，两部分的末端有2nt重叠，比如该读段的两部分分别为ATCGCTTAAG和AGTACGATTC，消除其负缺口即重叠的AG，获得对应的读段为ATCGCTTAAGTACGATTC。
在本发明的一个实施例中，本发明的一方面的方法中的作比对包括：将每对读段对的左臂和右臂分别与所述参考序列比对，获得一级左比对结果和一级右比对结果；分别以所述一级左比对结果和所述一级右比对结果的其中一个为参考，对另一个作比对，获得二级左比对结果和二级右比对结果；基于所述二级左比对结果和所述二级右比对结果，获得多个所述读段对的比对结果，或者获得多个所述左臂的比对结果和多个所述右臂的比对结果。这样经过两次比对，可以获得读段对比对结果，在本发明的一个实施例中，第一次比对是与参考序列作全局比对，以该次左臂/右臂比对结果为基准对右臂/左臂比对结果进行的第二次比对为局部比对，这样，能够将分别来自二级左比对结果和二级右比对结果中的比对到同一染色体上且之间的距离符合预期的两个读段配对成一对读段对，获得读段对比对结果。
在本发明的一个实施例中，所说的作比对包括，设置所述缺口的大小以使每个左臂或者每个右臂与所述参考序列进行多次比对，以获得最佳比对结果。例如，将所述每个左臂或者所述每个右臂的缺口分别设置为-3nt、-2nt、-1nt、0nt、1nt、2nt、3nt、4nt、5nt、6nt和7nt，获得对应的多个读段，分别将所述对应的多个读段与所述参考序列比对，将最优比对的序列作为该左臂/右臂，这里对于比对结果的好坏可以基于所利用的比对软件对比对结果的默认评判。
在本发明的一个实施例中，执行所述数据处理程序还包括实现，在所述消除比对结果中的每个读段的缺口之前，提取所述比对结果中的唯一比对结果以替换所述比对结果，所述唯一比对结果包括唯一比对上所述参考序列的多个读段对，并且每一所述读段对比对到所述参考序列的相同染色体，每一所述读段对中的两个读段的距离符合预期的其来自的所述染色体片段的两个位置之间的距离。
在本发明的一个实施例中，执行所述数据处理程序还包括实现，修正使所述唯一比对结果中的每一对读段对比对到所述参考序列的相同染色体的正链。例如，对于分别比对上一染色体的正负链的一对读段，将比对上负链的读段变成其反向互补链，这样以其反向互补链来替代该读段得以实现所说的修正。
在本发明的一个实施例中，执行所述数据处理程序还包括实现数据格式转换，所述数 据格式转换包括转换所述比对结果或所述唯一比对结果的格式。在本发明得一个实施利中，要求通用比对结果的格式为SAM或BAM，以利于后续的基于比对结果或比对结果对数据进一步分析，SAM或BAM是常见的二进制格式，BAM是SAM的压缩格式。由于利用不同的比对软件，可能输出的比对结果或者唯一比对结果的格式不适用于现有的后续数据处理或者分析软件程序，例如前述的TeraMap格式的比对结果，其输出的数据格式不满足现有的大部分变异检测软件SOAPsnp、GATK或SOAPindel等对输入数据格式的要求，转换数据格式使获得具有通用数据格式的通用比对结果，便于对比对数据进一步分析处理。
依据本发明的第二方面，提供一种测序数据处理系统，其包括一主机和一显示器，该系统还包括本发明一方面或者任一具体实施方式中的测序数据处理装置。前述对测序数据处理装置的优点及技术特征的描述，同样适用本发明的这一系统，在此不再赘述。
依据本发明的第三方面，提供一种测序数据处理方法，该方法包括如下步骤：获取测序数据，所述测序数据包括多对读段对，每对读段对由两个读段组成，分别来自一条染色体片段的两个位置，每对读段对中的两个读段分别来自所述染色体片段的正链和负链，或者每对读长对中的两个读段都来自所述染色体片段的正链或所述染色体片段的负链，每个读段都包含缺口，将一对读段对的两个读段分别定义为左臂和右臂；将所述测序数据与参考序列比对，获得比对结果，所述比对结果包括多个所述读段对的比对结果，和/或，所述比对结果包括多个所述左臂的比对结果和多个所述右臂的比对结果；消除所述比对结果中的每个读段的缺口，获得通用比对结果。关于读段对的获取方式、读段包含的缺口、比对、消除缺口，比对结果和通用比对结果等的特点可以参照上述对本发明一方面或者任一具体实施方式中的装置中的相应技术特征的描述。例如，同样的，这里所说的分别来自一条染色体片段的两个位置的读段对，可以通过构建末端文库(pair-end library)或者配对末端文库(mate-pair library)，对所构建的文库进行测序来获得，在本发明的一个实施例中，利用Complete Genomics公司(CG)的文库构建方法及其测序平台，获得多对读段对，一对读段对之间的距离是由读段的长度以及酶的识别位点和切割位点的距离来控制的。CG平台通过酶切构建多接头配对末端文库，利用特有的组合探针连接测序(cPAL)技术对所构建的环状文库进行测序，测读出接头两旁的碱基，因为其是利用酶切连接一个接头的两段来进行配对末端文库构建的，由于每一种酶都有一个首选的切割距离，而在实际酶切时经常比首选距离多一个位置或少一个位置，这样使得读段中经常带有缺口(gap)，缺口常为+1或者-1，和/或，建库时倘若使用同一种酶多次酶切，每次的酶切位置易发生变化，酶切位置的变化也会使获得的读段带有缺口，例如在构建多接头环状文库时，利用Alu酶两次酶切来连接多个接头的不同部分，读测这些接头旁的碱基时，会产生带+3/-3的缺口的读段。在本发明中缺口的大小还可以是0。来自多接头文库中的多个读段，一个读段可以和任一其 它读段组成一对读段对。这里所说的“正链”和“负链”是组成染色体片段的互补的两条链，是相对的，称一条链为正链就可以称其互补链为负链，在本发明的一个实施例中，将与参考序列匹配的链称为正链，将另一条链称为负链。在本发明中，比对可以利用已知比对软件进行，比如SOAP、BWA等，也可以利用CG平台的比对软件TeraMap进行。在本发明的一个实施例中，比对是利用TeraMap进行的，所得的比对结果的格式为TeraMap。在本发明的一个实施例中，所说的消除比对结果中每个读段的缺口是指，对带负缺口的读段去除掉其负缺口即去除掉重叠的碱基，对带正缺口的读段以N替代正缺口的大小，N为A、T、C或G，例如，对带负缺口比如为-2nt的读段，基于缺口处该读段可分成两部分，两部分的末端有2nt重叠，比如该读段的两部分分别为ATCGCTTAAG和AGTACGATTC，消除其负缺口即重叠的AG，获得对应的读段为ATCGCTTAAGTACGATTC。
在本发明的一个实施例中，获取所述测序数据包括构建测序文库，获得测序文库，所述测序文库为单链环状DNA文库，所述测序文库由所述染色体片段的一条链和至少一个预定DNA序列构成。所说的单链环状文库可以利用已知文库构建方法来构建，比如参考Life Technologies公司的SOLiD的配对末端文库的构建获得单接头环状双链文库，接着分离双链获得单链环状文库，在本发明的一个实施例中，单链环状文库是利用CG的建库技术来构建，文库构建可参考US7897344，获得多接头单链环状文库。
在本发明的一个实施例中，所述每对读段分别来自所述染色体片段的两端。通过参考改进CG的建库技术，利用一个接头的两部分分别连接于一段染色体片段的两端，单链分离，单链成环，获得1-接头单链环状文库，该1-接头单链环状文库由所述染色体片段的一条链和连接所述一条链的两端的一个预定DNA序列构成，滚环扩增形成DNA纳米球(DNB)，CG平台测序采用的是其独有的高密度DNA纳米芯片技术，其原理是在芯片上嵌入DNA纳米球，用非连续、非连锁联合探针锚定连续(cPAL)技术读取序列，关于DNB嵌入到芯片以及cPAL技术可分别参考US8278039B2和US8518640B2。所说的预定DNA序列为已知序列，为前述的接头或者接头的一条链。所说的改进CG建库方法构建1-接头环状单链文库包括步骤：(1)提取待测核酸；(2)末端磷酸化所述核酸，获得末端磷酸化产物；(3)末端修复所述末端磷酸化产物，获得末端修复产物；(4)将第一序列和第二序列连接至所述末端修复产物的两端，获得第一连接产物；(5)利用第三序列对所述连接产物进行缺刻平移和扩增，获得扩增产物，所述第三序列为一对引物对，所述引物对的至少一条引物带有生物素标记；(6)利用所述生物素标记对所述扩增产物进行单链分离，获得单链产物；(7)利用第四序列环化所述单链产物，获得所述测序文库；其中，所述第四序列能够连接所述第一序列的一端和所述第二序列的一端，所述第一序列和/或所述第二序列的另一端为双脱氧核苷酸。所说的第四序列能够连接所说的第一序列和所说的第二序列形 成一个所说的接头，缺刻平移是为消除连接在末端修复产物两端的第一序列和/或第二序列的另一端的双脱氧核苷酸造成的缺刻(nick)，利用至少一条引物带有生物素标记使扩增产物的至少一条链带有生物素标记，使后续易于基于该生物素标记分离获得单链产物。在本发明的一个实施例中，所说的改进CG建库方法构建1-接头环状单链文库包括步骤：(1)提取待测核酸；(2)末端修复所述核酸，获得末端修复产物；(3)末端磷酸化所述末端修复产物，获得末端磷酸化产物；(4)将第一序列和第二序列连接至所述末端磷酸化产物的两端，获得第一连接产物；(5)利用第三序列对所述连接产物进行缺刻平移和扩增，获得扩增产物，所述第三序列为一对引物对，所述引物对的至少一条引物带有生物素标记；(6)利用所述生物素标记对所述扩增产物进行单链分离，获得单链产物；(7)利用第四序列环化所述单链产物，获得所述测序文库；其中，所述第四序列能够连接所述第一序列的一端和所述第二序列的一端，所述第一序列和/或所述第二序列的另一端为双脱氧核苷酸。末端修复和末端磷酸化哪个步骤先进行不作限制。末端修复是为获得平末端核酸片段，使得能够连接其它核苷酸或序列。末端磷酸化是为了减少样本核酸片段的互相连接，使得核酸含量很低的样本也能够进行文库构建且满足文库上机量要求。单接头环状单链文库如图1所示，将所构建的单接头环状单链文库(1-AD)上机测序，1-AD测序输出读段对总长约为30bp，其中一读段12bp，一读段19bp，一读段对两个读段之间在基因组上的距离的中位数约为140bp。单接头建库量较小，适合cfDNA含量较少的情况，此外还有建库时间短及建库成本低的优点。
在本发明的一个实施例中，本发明的这一方法中的比对包括：将每对读段对的左臂和右臂分别与所述参考序列比对，获得一级左比对结果和一级右比对结果，分别以所述一级左比对结果和所述一级右比对结果的其中一个为参考，对另一个作比对，获得二级左比对结果和二级右比对结果，基于所述二级左比对结果和所述二级右比对结果，获得多个所述读段对的比对结果，或者获得多个所述左臂的比对结果和多个所述右臂的比对结果。这样经过两次比对，可以获得读段对比对结果，在本发明的一个实施例中，第一次比对是与参考序列作全局比对，以该次左臂/右臂比对结果为基准对右臂/左臂比对结果进行的第二次比对为局部比对，这样，能够将分别来自二级左比对结果和二级右比对结果中的比对到同一染色体上且之间的距离符合预期的两个读段配对成一对读段对，获得读段对比对结果。
在本发明的一个实施例中，所说的比对包括，设置所述缺口的大小以使每个左臂或者每个右臂与所述参考序列进行多次比对，以获得最佳比对结果。例如，将所述每个左臂或者所述每个右臂的缺口分别设置为-3nt、-2nt、-1nt、0nt、1nt、2nt、3nt、4nt、5nt、6nt和7nt，获得对应的多个读段，分别将所述对应的多个读段与所述参考序列比对，将最优比对的序列作为该左臂/右臂，这里对于比对结果的好坏可以基于所利用的比对软件对比对结果 的默认评判。
在本发明的一个实施例中，执行所述数据处理程序还包括实现，在所述消除比对结果中的每个读段的缺口之前，提取所述比对结果中的唯一比对结果以替换所述比对结果，所述唯一比对结果包括唯一比对上所述参考序列的多个读段对，并且每一所述读段对比对到所述参考序列的相同染色体，每一所述读段对中的两个读段的距离符合预期的其来自的所述染色体片段的两个位置之间的距离。
在本发明的一个实施例中，执行所述数据处理程序还包括实现，修正使所述唯一比对结果中的每一对读段对比对到所述参考序列的相同染色体的正链。例如，对于分别比对上一染色体的正负链的一对读段，将比对上负链的读段变成其反向互补链，这样以其反向互补链来替代该读段得以实现所说的修正。
在本发明的一个实施例中，执行所述数据处理程序还包括实现数据格式转换，所述数据格式转换包括转换所述比对结果或所述唯一比对结果的格式。在本发明得一个实施利中，要求通用比对结果的格式为SAM或BAM，以利于后续的基于比对结果或比对结果对数据进一步分析，SAM或BAM是常见的二进制格式，BAM是SAM的压缩格式。由于利用不同的比对软件，可能输出的比对结果或者唯一比对结果的格式不适用于现有的后续数据处理或者分析软件程序，例如前述的TeraMap格式的比对结果，其输出的数据格式不满足现有的大部分变异检测软件SOAPsnp、GATK或SOAPindel等对输入数据格式的要求，转换数据格式使获得具有通用数据格式的通用比对结果，便于对比对数据进一步分析处理。
依据本发明的第四方面，本发明提供一种计算机可读存储介质，其用于存储供计算机执行的程序，所述程序的执行包括完成前述本发明一方面的或者其任一具体实施方式中的测序数据处理方法。前述对本发明的测序数据处理方法的优点和技术特征的描述也适用于该计算机可读存储介质，在此不再赘述。所称存储介质可以包括：只读存储器、随机存储器、磁盘或光盘等。
依据本发明的第五方面，本发明提供一种检测单核苷酸多态性(SNP)的方法，该方法包括：A.获取待测样本的核酸；B.对所述核酸的至少一部分进行测序，获得测序数据；C.对所述测序数据进行处理，以获得通用比对结果；D.基于所述通用比对结果检测SNP；其中，C步利用骤本发明一方面的或者任一具体实施方式中的测序数据处理装置和/或方法进行。上述对本发明的测序数据处理装置和/或方法的优点及技术特征的描述，也适用于本发明这一方面的SNP检测方法，在此不再赘述。
在本发明的一个实施例中，B步骤包括，对所述核酸的至少一部分进行测序文库构建，获得测序文库，所述测序文库为单链环状DNA文库，所述单链环状DNA文库的构建包括：末端磷酸化所述核酸，获得末端磷酸化产物；末端修复所述末端磷酸化产物，获得末端修 复产物；将第一序列和第二序列连接至所述末端修复产物的两端，获得第一连接产物；利用第三序列对所述连接产物进行缺刻平移和扩增，获得扩增产物，所述第三序列为一对引物对，所述引物对的至少一条引物带有生物素标记；利用所述生物素标记对所述扩增产物进行单链分离，获得单链产物；环化所述单链产物，获得所述测序文库；其中，所述第四序列能够连接所述第一序列的一端和所述第二序列的一端，所述第一序列和/或所述第二序列的另一端为双脱氧核苷酸。在本发明的另一实施例中，先进行末端修复再进行末端磷酸化。末端修复是为获得平末端核酸片段，使得能够连接其它核苷酸或序列。末端磷酸化是为了减少样本核酸片段的互相连接，使得核酸含量很低的样本也能够进行文库构建且满足文库上机量要求。所说的第四序列能够连接第一序列和第二序列形成一个所说的接头，缺刻平移是为消除连接在末端修复产物两端的第一序列和/或第二序列的另一端的双脱氧核苷酸造成的缺刻(nick)，利用至少一条引物带有生物素标记使扩增产物的至少一条链带有生物素标记，使后续易于基于该生物素标记分离获得单链产物。单接头环状单链文库如图1所示，单接头建库量较小，适合cfDNA含量较少的情况，此外还有建库时间短及建库成本低的优点。在本发明的一个实施例中，对构建的文库进行测序是利用组合探针锚定连接测序技术进行的，例如利用CG测序平台进行，CG测序可避免测序中错误的积累，其准确性较合成法和连接法测序高，可达99.999％，通过CG平台单接头测序获得测序数据，成本更低速度也更快。
基于通用比对结果检测SNP可以利用目前已知的SNP检测方法和/或软件，比如利用SOAP2、samtools、GATK等。在本发明的一个实施例中，使用开源软件samtools对通用比对结果(bam文件)进行预处理，包括排序、去除由于建库PCR产生的重复reads等，之后使用samtools mpileup和/或GATK开源软件得到SNP结果，并用开源软件bcftools转换为vcf格式。相比其他工具Samtools操作简单，输出格式通用，在大数据处理过程中可以使用多线程提高效率。
依据本发明的第六方面，本发明提供一种SNP检测设备，该设备用以执行本发明一方面的SNP检测方法的全部或部分步骤，所述设备包括：核酸获取装置，用以获取待测样本的核酸；测序装置，用以对来自所述核酸获取单元的核酸的至少一部分进行测序，获得测序数据，所述测序数据包括多对读段对，每对读段对由两个读段组成，分别来自一条染色体片段的两个位置，每对读长对中的两个读段分别来自所述染色体片段的正链和负链，或者每对读长对中的两个读段都来自所述染色体片段的正链或所述染色体的负链，每个读段都包含缺口，将一对读段对的两个读段分别定义为左臂和右臂；数据处理装置，用于对来自所述测序装置的测序数据进行处理，以获得通用比对结果；检测装置，用于基于来自所述数据处理装置的通用比对结果检测所述SNP；其中，所述数据处理装置包括，数据接收 单元，用于接收来自所述测序装置的测序数据，处理器，用于执行数据处理程序，执行所述数据处理程序包括实现将所述测序数据与参考序列作比对，获得比对结果，以及消除所述比对结果中的每个读段的缺口，获得通用比对结果，所述比对结果包括多个所述读段对的比对结果，和/或，所述比对结果包括多个所述左臂的比对结果和多个所述右臂的比对结果，以及，至少一个存储单元，用于存储数据，其中包括所述数据处理程序。前述对本发明一方面的或者任一其具体实施方式中的SNP检测方法的优点和技术特征的描述，同样适用本发明这一方面的SNP检测设备，在此不再赘述，而且，本领域普通技术人员可以理解，本发明的这一装置中的全部或部分单元，可选择的、可拆卸的包含一个或多个子单元以执行或实现前述本发明SNP检测方法的各个具体实施方式。
利用本发明的数据处理装置、系统和/或方法，包括开发TeraMap2Sam转换软件，准确处理掉测序数据中的gap、将CG平台TeraMap的比对结果转化为通用的SAM格式，使后续能够直接使用Samtools，GATK等众多优秀的开源软件进行变异检测，使后续分析的选择更加广泛。利用本发明的SNP检测方法和/或设备，包括使用samtools软件做SNP分析简单通用、速度快且可信度高。
附图说明
本发明的上述和/或附加的方面和优点从结合下面附图对实施方式的描述中将变得明显和容易理解，其中：
图1是本发明的一个实施例中的单接头环状单链文库的结构示意图；
图2是本发明的一个实施例中的测序数据处理装置的结构示意图；
图3是本发明的一个实施例中的测序数据处理系统的结构示意图；
图4是本发明的一个实施例中的测序数据处理方法的流程图；
图5是本发明的一个实施例中的测序数据处理方法的流程图；
图6是本发明的一个实施例中的SNP检测方法的流程图；
图7是本发明的一个实施例中的SNP检测设备的结构示意图；
图8是本发明的一个实施例中的单接头文库构建和测序流程图；
图9是本发明的一个实施例中的SNP检测流程图；
图10是本发明的一个实施例中的Teramap2Sam软件的算法流程图。
具体实施方式
下面详细描述本发明的实施例，所述实施例的示例在附图中示出，其中，自始至终相同或类似的标号表示相同或类似的元件或具有相同或类似功能的元件。下面通过参考附图 描述的实施例是示例性的，仅用于解释本发明，而不能理解为对本发明的限制。需要说明的是在本文中所使用的术语“第一”、“第二”、“第三”、“第四”或者“一级”、“二级”等仅为方便描述指代，而不能理解为指示或暗示相对重要性，也不能理解为之间有先后顺序关系。在本发明的描述中，除非另有说明，“多个”的含义是两个或两个以上。
图2显示本发明的测序数据处理装置的一个实施例的装置结构示意图，该测序数据处理装置100包括：数据接收单元10、处理器20和一个存储单元30，处理器20与数据接收单元10和存储单元30连接，存储单元30和数据处理单元10连接。数据接收单元10，用于接收测序数据，所述测序数据包括多对读段对，每对读段对由两个读段组成，分别来自一条染色体片段的两个位置，每对读长对中的两个读段分别来自所述染色体片段的正链和负链，或者每对读段对中的两个读段都来自所述染色体片段的正链或所述染色体的负链，每个读段都包含缺口，将一对读段对的两个读段分别定义为左臂和右臂。这里所说的分别来自一条染色体片段的两个位置的读段对，可以通过构建末端文库(pair-end library)或者配对末端文库(mate-pair library)，对所构建的文库进行测序来获得，在本发明的一个实施例中，利用Complete Genomics公司(CG)的文库构建方法及其测序平台，获得多对读段对，一对读段对之间的距离是由读段的长度以及酶的识别位点和切割位点的距离来控制的。CG平台通过酶切构建多接头配对末端文库，利用特有的组合探针连接测序(cPAL)技术对所构建的环状文库进行测序，测读出接头两旁的碱基，因为其是利用酶切连接一个接头的两段来进行配对末端文库构建的，由于每一种酶都有一个首选的切割距离，而在实际酶切时经常比首选距离多一个位置或少一个位置，这样使得读段中经常带有缺口(gap)，缺口常为+1或者-1，和/或，建库时倘若使用同一种酶多次酶切，每次的酶切位置易发生变化，酶切位置的变化也会使获得的读段带有缺口，例如在构建多接头环状文库时，利用Alu酶两次酶切来连接多个接头的不同部分，读测这些接头旁的碱基时，会产生带+3/-3的缺口的读段。在本发明中缺口的大小还可以是0。以CG平台目前的双接头(two adaptors，2-AD)测序文库为例，2-AD测序输出总长为60bp，可分为两对读段对(mate-paired reads)，每对读段对中的读段在10bp的位置都有小的gap，在20bp位置有一个无效测序位点N，一对读段对的两个读段之间的在基因组上的距离一般小于2000bp。来自多接头文库中的多个读段，一个读段可以和任一其它读段组成一对读段对。这里所说的“正链”和“负链”是组成染色体片段的互补的两条链，是相对的，称一条链为正链就可以称其互补链为负链，在本发明的一个实施例中，将与参考序列匹配的链称为正链，将另一条链称为负链。
处理器20，用于执行数据处理程序，执行所述数据处理程序包括实现将所述测序数据与参考序列作比对，获得比对结果，以及消除所述比对结果中的每个读段的缺口，获得通用比对结果，所述比对结果包括多个所述读段对的比对结果，和/或，所述比对结果包括多 个所述左臂的比对结果和多个所述右臂的比对结果。比对可以利用已知比对软件进行，比如SOAP、BWA等，也可以利用CG平台的比对软件TeraMap进行。在本发明的一个实施例中，比对是利用TeraMap进行的，所得的比对结果的格式为TeraMap。在本发明的一个实施例中，所说的消除比对结果中每个读段的缺口是指，对带负缺口的读段去除掉其负缺口即去除掉重叠的碱基，对带正缺口的读段以N替代正缺口的大小，N为A、T、C或G，对缺口为0的读段不作处理，例如，对带负缺口比如为-2nt的读段，基于缺口处该读段可分成两部分，两部分的末端有2nt重叠，比如该读段的两部分分别为ATCGCTTAAG和AGTACGATTC，消除其负缺口即重叠的AG，获得对应的读段为ATCGCTTAAGTACGATTC。
存储单元30，用于存储数据，存储单元30中存储有上述数据处理程序，也存储来自数据接收单元10的测序数据和处理器20的处理的中间数据或结果。
图3显示本发明的测序数据处理系统的一个实施例中的系统结构示意图。该测序数据处理系统1000包括：测序数据处理装置100，主机200和显示装置300。主机200可为一音频/视频/信号发送源装置，比如电脑主机、大型机等，其用以传送显示装置300所需的显示数据。该主机200包含至少一个与测序数据处理装置100电性连接的接口，测序数据处理装置100接收从主机200输出的测序数据，并对测序数据进行处理，然后将处理的数据或结果输出到显示装置300。
图4显示本发明的测序数据处理方法的一个实施例的测序数据处理流程图。该测序数据处理方法包括步骤：S1获取测序数据，所述测序数据包括多对读段对，每对读段对由两个读段组成，分别来自一条染色体片段的两个位置，每对读段对中的两个读段分别来自所述染色体片段的正链和负链，或者每对读长对中的两个读段都来自所述染色体片段的正链或所述染色体片段的负链，每个读段都包含缺口，将一对读段对的两个读段分别定义为左臂和右臂；S2将所述测序数据与参考序列比对，获得比对结果，所述比对结果包括多个所述读段对的比对结果，和/或，所述比对结果包括多个所述左臂的比对结果和多个所述右臂的比对结果；S3消除所述比对结果中的每个读段的缺口，获得通用比对结果。关于读段对的获取方式、读段包含的缺口、比对、消除缺口，比对结果和通用比对结果等的特点可以参照上述对本发明一方面或者任一具体实施方式中的测序数据处理装置中的相应技术特征的描述。例如，同样的，这里所说的分别来自一条染色体片段的两个位置的读段对，可以通过构建末端文库(pair-end library)或者配对末端文库(mate-pair library)，对所构建的文库进行测序来获得，在本发明的一个实施例中，利用Complete Genomics公司(CG)的文库构建方法及其测序平台，获得多对读段对，一对读段对之间的距离是由读段的长度以及酶的识别位点和切割位点的距离来控制的。CG平台通过酶切构建多接头配对末端文库，利 用特有的组合探针连接测序(cPAL)技术对所构建的环状文库进行测序，测读出接头两旁的碱基，因为其是利用酶切连接一个接头的两段来进行配对末端文库构建的，由于每一种酶都有一个首选的切割距离，而在实际酶切时经常比首选距离多一个位置或少一个位置，这样使得读段中经常带有缺口(gap)，缺口常为+1或者-1，和/或，建库时倘若使用同一种酶多次酶切，每次的酶切位置易发生变化，酶切位置的变化也会使获得的读段带有缺口，例如在构建多接头环状文库时，利用Alu酶两次酶切来连接多个接头的不同部分，读测这些接头旁的碱基时，会产生带+3/-3的缺口的读段。在本发明中缺口的大小还可以是0。来自多接头文库中的多个读段，一个读段可以和任一其它读段组成一对读段对。这里所说的“正链”和“负链”是组成染色体片段的互补的两条链，是相对的，称一条链为正链就可以称其互补链为负链。这里，将与参考序列匹配的链称为正链，将另一条链称为负链。比对可以利用已知比对软件进行，比如SOAP、BWA等，也可以利用CG平台的比对软件TeraMap进行。在本发明的一个实施例中，比对是利用TeraMap进行的，所得的比对结果的格式为TeraMap。在本发明的一个实施例中，所说的消除比对结果中每个读段的缺口是指，对带负缺口的读段去除掉其负缺口即去除掉重叠的碱基，对带正缺口的读段以N替代正缺口的大小，N为A、T、C或G，对缺口为0的读段不作处理，例如，对带负缺口比如为-2nt的读段，基于缺口处该读段可分成两部分，两部分的末端有2nt重叠，比如该读段的两部分分别为ATCGCTTAAG和AGTACGATTC，消除其负缺口即重叠的AG，获得对应的读段为ATCGCTTAAGTACGATTC。
图5是本发明的测序数据处理方法的一个实施例的数据处理流程图。该测序数据处理方法包括：S10获取测序数据，所述测序数据包括多对读段对，每对读段对由两个读段组成，分别来自一条染色体片段的两个位置，每对读段对中的两个读段分别来自所述染色体片段的正链和负链，或者每对读长对中的两个读段都来自所述染色体片段的正链或所述染色体片段的负链，每个读段都包含缺口，将一对读段对的两个读段分别定义为左臂和右臂；S20将所述测序数据与参考序列比对，获得比对结果，所述比对结果包括多个所述读段对的比对结果，和/或，所述比对结果包括多个所述左臂的比对结果和多个所述右臂的比对结果；S30提取所述比对结果中的唯一比对结果以替换所述比对结果，所述唯一比对结果包括唯一比对上所述参考序列的多个读段对，并且每一所述读段对比对到所述参考序列的相同染色体，每一所述读段对中的两个读段的距离符合预期的其来自的所述染色体片段的两个位置之间的距离；S40修正使所述唯一比对结果中的每一对读段对比对到所述参考序列的相同染色体的正链。例如，对于分别比对上一染色体的正负链的一对读段，将比对上负链的读段变成其反向互补链，这样以其反向互补链来替代该读段得以实现所说的修正；S50消除所述唯一比对结果中的每个读段的缺口，获得通用比对结果。
图6是本发明的SNP检测方法的一个实施例的检测流程图。该SNP检测方法包括步骤：S11获取待测样本的核酸；S12对所述核酸进行测序，获得测序数据；S13对所述测序数据进行处理，以获得通用比对结果；S14基于所述通用比对结果检测所述SNP；其中，S13是利用本发明一方面的或者任一具体实施方式中的测序数据处理装置和/或测序数据处理方法进行的。基于通用比对结果检测SNP可以利用目前已知的SNP检测方法和/或软件工具，比如利用SOAP2、GATK、samtools等。
图7是本发明的SNP检测设备的一个实施例的设备结构示意图。该设备2000包括：核酸获取装置200，用以获取待测样本的核酸；测序装置400，用以对来自所述核酸获取单元的核酸进行测序，获得测序数据，所述测序数据包括多对读段对，每对读段对由两个读段组成，分别来自一条染色体片段的两个位置，每对读长对中的两个读段分别来自所述染色体片段的正链和负链，或者每对读长对中的两个读段都来自所述染色体片段的正链或所述染色体的负链，每个读段都包含缺口，将一对读段对的两个读段分别定义为左臂和右臂；数据处理装置600，用于对来自所述测序装置的测序数据进行处理，以获得通用比对结果；检测装置800，用于基于来自所述数据处理装置600的通用比对结果检测SNP；其中，所述数据处理装置600包括，数据接收单元610，用于接收来自所述测序装置的测序数据，处理器630，用于执行数据处理程序，执行所述数据处理程序包括实现将所述测序数据与参考序列作比对，获得比对结果，以及消除所述比对结果中的每个读段的缺口，获得通用比对结果，所述比对结果包括多个所述读段对的比对结果，和/或，所述比对结果包括多个所述左臂的比对结果和多个所述右臂的比对结果，以及，至少一个存储单元650，用于存储数据，其中包括所述数据处理程序。前述对本发明一方面的或者任一其具体实施方式中的SNP检测方法的优点和技术特征的描述，同样适用本发明这一方面的SNP检测设备，在此不再赘述，而且，本领域普通技术人员可以理解，本发明的这一装置中的全部或部分单元，可选择的、可拆卸的包含一个或多个子单元以执行或实现前述本发明SNP检测方法的各个具体实施方式。
以下实施例仅用于说明本发明的优选实施方式，实施例中未注明具体操作手段或条件的，可以按照本领域内的文献所描述的技术或条件(例如参考J.萨姆布鲁克等著，黄培堂等译的《分子克隆实验指南》，第三版，科学出版社)或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品或服务。
实施例一
以下以肺癌患者的外周血血浆作为检测对象，样本来自西南医院，进行如下检测：
(一)文库建立及测序
建库及测序流程如图8所示，以下涉及的具体序列，都是从左到右为5’端至3’端，序列中的“//”中为末端修饰基团，“phos”表示磷酸化，“dd”表示双脱氧，“bio”表示生物素。
1、cfDNA的提取(采用SnoMag Circulating DNA Kit)：
1)取200ul血浆于1.5mlEP管，加入600ul buffer LSB。
2)加入20μlNanoMag Circulating Beads混匀，室温放置10min，每2-3min混匀一次。
3)将EP管置于磁力架上吸附1min，弃上清。
4)取下EP管加入150uL Buffer WA，混匀。
5)将EP管置于磁力架上吸附1min，弃上清。
6)取下EP管加入150uL 75％乙醇，混匀。
7)将EP管置于磁力架上吸附1min，弃上清。
8)重复6-7一次。
9)室温干燥磁珠5min。
10)加入32ul elution buffer混匀磁珠，室温静置5min。
11)将EP管置于磁力架上吸附1min，转移上清至新的1.5mlEP管。
2、文库的构建：
1)rSAP去磷酸化反应
cfDNA 30ul
10x NEBuffer 2 3.5ul
rSAP(1U/ul) 1.5ul
Total 35ul
反应条件：
Figure PCTCN2014093516-appb-000002
2)T4DNA Polymerase末端补平
Figure PCTCN2014093516-appb-000003
反应条件：
12℃ 20min
4℃ hold
60ul AmpureXP beads纯化以上反应产物，22ulElution buffer洗脱。
3)第一序列和第二序列分别连接到末端补平的DNA片段的两端
Figure PCTCN2014093516-appb-000004
反应条件：
20℃ 15min
4℃ hold
40ul AmpureXPbeads纯化以上反应产物,22ulElution buffer洗脱。
第一序列的两条链为：TTGGCCTCCGACT/3-ddT/(SEQ ID NO：1)，
/5phos/AAGTCGGAGGCCAAGCGGTCGT/ddC/(SEQ ID NO：2)。
第二序列的两条链分别为：/5Phos/GTCTCCAGTCGAAGCCCGACG/3ddC/(SEQ ID NO：3)，GCTTCGACTGGAGA/3ddC/(SEQ ID NO：4)。
4)缺刻平移(Nick Translation)
Figure PCTCN2014093516-appb-000005
第三序列中的上游引物/5-bio/TCCTAAGACCGCTTGGCCTCCGACT(SEQ ID NO：5)，
第三序列中的下游引物
5Phos/AGACAAGCTCxxxxxxxxxxGATCGGGCTTCGACTGGAGAC(SEQ ID NO：6)，中间“x”处为可变的标签序列区域，可以以N替代，N为A、T、C或G，当没有其它样本文库一起混合上机，只有一个样本文库上机，不需要标签序列，即第三序列可为
5Phos/AGACAAGCTCGATCGGGCTTCGACTGGAGAC(SEQ ID NO：7)，在该示例中， 由于是肿瘤游离核酸样本，混合核酸中的目标核酸(ctDNA)含量低，若多个这样的样本文库混合上机获得混合数据，需要拆分混合数据对应到各自样本，会损失一部分数据，且构建的是单接头环状文库读段相对短，要准确检测变异需要深度测序获得相对大量的测定数据，较佳的，单个样本文库上机。
反应条件：
60℃ 5min
37℃ 0.1℃/secs-hold
向上步反应物中加入如下8ul Nick Translation mix
Figure PCTCN2014093516-appb-000006
反应条件：
37℃ 20min
4℃ hold
40ul AmpureXP beads纯化以上反应产物,37.4ulElution buffer洗脱。
5)PCR with Pfx
Figure PCTCN2014093516-appb-000007
反应条件：
Figure PCTCN2014093516-appb-000008
50ul AmpureXP beads纯化以上反应产物，22ulElution buffer洗脱。
6)Qubit定量
利用Qubit dsDNA HS assay kit对PCR产物进行浓度测定。
7)链分离(Strand Separation)
a)多个文库混合，使DNA共约160ng。样品补1xTE至总体积为60ul。
b)提前准备以下试剂：4X BBB，Streptavidin Beads，0.3M MOPS acid，0.5％Tween20，1X BBB/Tween Mix，1X BWB/Tween Mix，0.1M NaOH。其中1X BWB/Tween Mix、0.1M NaOH，Streptavidin Beads需现配现用。
c)提前15min配置以下四种试剂
0.5％Tween20，1X BBB/Tween Mix，1X BWB/Tween Mix，0.1M NaOH.
其中0.5％Tween20配置方法同前述，其他三种配置方法如下：
d)1X BBB/Tween Mix
1X BBB 30ul
0.5％Tween20 0.3ul
Total 30.3ul
e)1X BWB/Tween Mix
1X BWB 2000ul
0.5％Tween20 20ul
Total 2020ul
f)0.1M NaOH
0.5M NaOH 15.6ul
Water 62.40ul
Total 78.0ul
g)Streptavidin Beads洗涤方法如下：
·每个样品取30ul Streptavidin Beads：加入3-5倍体积的1XBBB，混匀后置于磁力架上静止吸附，调整不粘管的方向，使得beads在1XBBB洗液中前后游动，弃上清液后，重复上述操作一次，
·取出不黏管加入1倍体积(30ul)1X BBB/Tween Mix悬浮，混匀后室温静置。
h)向60ulPCR产物样品中加入20ul 4XBBB混匀，然后转移到上步骤含有30ul1X BBB/Tween Mix溶解的beads的不粘管中混匀，此110ul混合物室温下结合15-20min，中间轻轻弹匀一次。
i)将上述不粘管磁力架放置3-5min，弃去上清液，用1ml的1X BWB/Tween Mix洗涤2次，方法同Streptavidin Beads的洗涤方法
j)向上述beads中加入26ul 0.1M NaOH，吹打混匀后放置10min，再置于磁力架上3-5min，取上清到新的1.5ml EP管中。
k)向上述1.5mlEP管中加入13ul 0.3M MOPS，混匀备用。
l)此步骤产物可以冻存于-20℃。
8)环化(Splint Circulation)
a)向上一步得到的39ul的样品中加入10ul的20uM第四序列，第四序列为
TCGAGCTTGTCTTCCTAAGACCGC(SEQ ID NO：8)；
b)提前5分钟准备连接酶反应混合液，配制如下：
Water 4.2ul
10x TA Buffer(LK1) 6ul
100mM ATP 0.6ul
600U/ul Ligase 0.2ul
Total 11ul
c)将连接酶反应混合液震荡充分混匀，离心后，向已经加入引物反应混合液的EP管中加入连接酶反应混合液11ul，震荡10s混匀，瞬时离心。
d)置于PCR仪中37℃孵育1.5h。
e)反应完成后，取出5ul样品，待6％变性胶电泳检测，剩余的约55ul体积，进入下一步酶反应。
9)酶切消化(Exo I and III)
a)提前5分钟左右准备引物反应混合液，配制如下：
10x TA Buffer(LK1) 1ul
20U/ul Exo I 3ul
200/ul Exo III 1ul
Total 5ul
b)将上述混合液震荡充分混匀，离心后，向上一步得到的55ul的样品中分别加入5ul的反应混合液；
c)震荡10s混匀离心，置于PCR仪中37℃孵育30min。
d)酶切30min完成后，向样品中加入2.5ul 500mM EDTA终止酶反应。
e)上述样品用PEG32beads/tween20纯化，方法如下：
将上步骤样品59ul转移到1.5ml不粘管中，加入78ul的PEG32beads/tween20(PEG32beads：tween20＝100:1)，室温结合15min，期间吹打混匀一次；
f)不粘管置于磁力架3-5min后弃去上清，用700ul 75％乙醇洗涤两次，洗涤时将不粘管前后方向反转，使得beads在乙醇中游动，每次洗涤游动2-3次；
g)室温下晾干后用27ul TE/tween20回溶(TE:tween20＝500:1)，溶解时间共计15min，中间混匀一次；
h)上清转移到新的1.5mlEP管中，将最终得到产物用QubitTM ssDNA Assay Kit定量。Buffer与染料比例为199:1混匀后votex并离心混合备用，取两份190ul稀释后染料工作液分别加入10ul的两种标准品votex并离心混合备用，取198ul稀释后染料工作液加入2ul样品，votex后并离心进行Qubit仪器定量。
i)浓度标准化(Normalization)
按照单链分子定量测定的浓度调整DNB制备使用的样本起始量统一调整为35.3ng－53ng，将对应体积样本(<60ul)转移至Biorad PCR板中，使用1XTE补齐使总体积不超过120ul。
终浓度为5.625-7.5fmol/ul，体积为120ul，则总量为35.3ng-53ng，1adapter测序中的DNB需要120fmol，7.5foml/ul，16ul。故需要把文库稀释至7.5fmol/ul。
a)CG 1-Adapter测序
利用CG平台的标准化流程测序。DNA纳米芯片是CG独创的一种高通量测序技术。该示例的对改进的单接头测序文库进行测序，较其他测序方案成本更低、速度更快，并集成质控确保其测序质量。
实施例二
对实施例一的下机数据进行处理，图9是检测SNP的流程示意图。利用本发明的测序数据处理方法和/或SNP检测方法，基于CG平台测序技术，可以对超微量的cfDNA进行富集，文库建立、测序和数据分析工作。在该示例中，由于CG测序原理的特殊性，其测序的reads较短，且在特定位置存在重测序以及小gap的现象，难以不处理直接使用普通的比对软件对测序结果进行比对或者检测分析。针对reads的特殊结构，我们使用CG平台专有的TeraMap进行比对，其工作原理是：首先，它将读长中的两端(LeftArm，RightArm)分别做比对，其间TeraMap会尝试多种gap值来处理读长，以获得更多的比对结果；然后，将每一端的比对结果拿出来作为参考，对另一端做局部比对(例如4-AD，局部比对的范围是0～700bp)；如果两端可以良好比对到同一染色体，且insert-size符合期望(例如4-AD，一读段对的两读段的距离为0～700bp)，则只输出最佳比对结果，否则两端的多个比对结果全部输出。TeraMap是CG测序平台的比对软件，它可将CG特有序列比对到参考基因组上，其输出格式由三部分组成，简要说明如下：第一部分，第一行是reads序列信息，包含reads的编号，左臂和右臂序列三列；第二部分：第二行和第三行是是reads比对情况说明，是左臂和右臂的比对情况的简述，格式是“字段名＝值”；第三部分：第四行至下一条reads序列信息的开头，即第四行和第五行，是reads比对结果详细信息。
第一部分：
列号 字段 类型 简介
1 QNAME 字符串 参考序列编号
2 POS 整型 比对到参考序列的位置
3 SEQ 字符串 比对片段的序列信息
第二部分：
Figure PCTCN2014093516-appb-000009
第三部分：
Figure PCTCN2014093516-appb-000010
因为TeraMap比对存在gap问题，使得无法进行下游分析，依据本发明的方法开发Teramap2Sam软件，将TeraMap比对结果中gap去除并转换为SAM(sequence alignment/map format)。Teramap2Sam软件的主要流程可分为三部分，算法流程图如图10所示。
第一步：提取唯一比对结果。根据TeraMap输出结果matchCount判定是否唯一比对，同时要求插入片段长度满足要求以及两端read比对在用一条参考序列上。
第二步：去除gap。根据gaps字段判定reads中的gap位置，并修正read序列。
第三步：计算FLAG。根据双端read的比对方向，计算SAM文件中的FLAG参数，获得比对情况。
SAM是存储比对信息的一种较通用的格式，每一行是一个reads的比对结果，主要由十一个字段组成，其后还可添加更多字段包含更多信息，比如XT:A:U就是表示此reads为unique比对。简要说明如下：
列号 字段 类型 简介
1 QNAME 字符串 比对读段的编号
2 FLAG 整型 标识符之和，用于表述比对情况
3 RNAME 字符串 参考序列的编号
4 POS 整型 比对上的位置
5 MAPQ 整型 比对质量值
6 CIGAR 字符串 简要比对信息表达式
7 RNEXT 字符串 下一读段比对上的参考序列的编号
8 PNEXT 整型 下一读段比对上的位置
9 TLEN 整型 比对读段长度
10 SEQ 字符串 序列读段的序列信息
11 QUAL 字符串 序列的质量信息
在实际使用中为了节约存储资源，主要使用其二进制压缩格式(BAM)。此外CG又针对其reads结构开发了Assembly Software将reads重新组装，组装完成后进行变异检测分析等后续工作。
由于CG单接头reads的特殊结构存在reads太短(最短12bp)的缺点，在一些特殊数据的处理中CG原有的突变检测工具不再适用或者检测结果不佳。针对这种情况，我们首先开发工具将TeraMap的比对结果转化为通用的SAM/BAM格式，其中SAM/BAM是高通量测序中普遍使用的比对结果格式，所以我们采用这种通用的格式，然后再使用BAM数据检测SNP变异。SNP检测可以利用已知开源软件，比如利用SOAP2、samtools、GATK等。在该示例中使用开源软件samtools对通用比对结果(SAM格式的bam文件)进行预处理，包括排序、去除由于建库PCR产生的重复reads等，之后使用samtools mpileup和/或GATK开源软件得到SNP结果，并用开源软件bcftools转换为vcf格式。相比其他工具Samtools操作简单，输出格式通用，在大数据处理过程中可以使用多线程提高效率，速度快可信度高。
较之传统方法，我们使用的基于CG单接头测序的方法可以实现超微量建库测序，建库只需要1-10ng核酸，需要外周血量2-5ml，并且CG的标准化流程简单快速，TeraMap比对结果转换为SAM格式之后比闭源的TeraMap格式更加通用，可以使用Samtools等软件进行处理。该示例中利用TeraMap进行比对。测序完成后使用CG平台的集成工具makeADF得到原始reads，然后用TeraMap进行比对，将测序得到的reads比对的参考序列上。得到的比对结果使用TeraMap2Sam转换为通用的SAM格式，接着利用Samtools等软件进行SNP检测，结果如表1。
表1
BAM(排序并去重) 198Gb/4h20min
SNP(vcf) 3.1Gb/2h38min
测序深度 85.62X
1X深度以上覆盖度 91.66％
5X深度以上覆盖度 89.97％
10X深度以上覆盖度 87.85％
1X深度以上CDS区域覆盖度 99.67％
5X深度以上CDS区域覆盖度 98.37％
10X深度以上CDS区域覆盖度 96.04％
总共检测到的肿瘤体细胞SNP数量 26533

Claims (33)

  1. 一种测序数据处理装置，其特征在于，包括，
    数据接收单元，用于接收所述测序数据，所述测序数据包括多对读段对，每对读段对由两个读段组成，分别来自一条染色体片段的两个位置，每对读段对中的两个读段分别来自所述染色体片段的正链和负链，或者每对读长对中的两个读段都来自所述染色体片段的正链或所述染色体的负链，每个读段都包含缺口，将一对读段对的两个读段分别定义为左臂和右臂；
    处理器，用于执行数据处理程序，执行所述数据处理程序包括实现将所述测序数据与参考序列作比对，获得比对结果，以及消除所述比对结果中的每个读段的缺口，获得通用比对结果，所述比对结果包括多个所述读段对的比对结果，和/或，
    所述比对结果包括多个所述左臂的比对结果和多个所述右臂的比对结果；以及，
    至少一个存储单元，用于存储数据，其中包括所述数据处理程序。
  2. 权利要求1的装置，其特征在于，所述作比对包括，
    将每对读段对的左臂和右臂分别与所述参考序列比对，获得一级左比对结果和一级右比对结果，
    分别以所述一级左比对结果和所述一级右比对结果的其中一个为参考，对另一个作比对，获得二级左比对结果和二级右比对结果，
    基于所述二级左比对结果和所述二级右比对结果，获得多个所述读段对的比对结果，或者获得多个所述左臂的比对结果和多个所述右臂的比对结果。
  3. 权利要求2的装置，其特征在于，所述作比对包括，设置所述缺口的大小以使每个左臂或者每个右臂与所述参考序列进行多次比对。
  4. 权利要求3的装置，其特征在于，所述每个左臂或者每个右臂与参考序列进行多次比对为，将所述每个左臂或者所述每个右臂的缺口分别设置为-3nt、-2nt、-1nt、0nt、1nt、2nt、3nt、4nt、5nt、6nt和7nt，获得对应的多个读段，分别将所述对应的多个读段与所述参考序列比对。
  5. 权利要求1-4任一装置，其特征在于，所述比对结果的格式为TeraMap。
  6. 权利要求1-5任一装置，其特征在于，执行所述数据处理程序还包括实现，在所述消除比对结果中的每个读段的缺口之前，提取所述比对结果中的唯一比对结果以替换所述比对结果，所述唯一比对结果包括唯一比对上所述参考序列的多个读段对，并且每一所述读段对比对到所述参考序列的相同染色体，每一所述读段对中的两个读段的距离符合所述染色体片段的两个位置的距离。
  7. 权利要求6的装置，其特征在于，执行所述数据处理程序还包括实现，修正使所述唯一比对结果中的每一对读段对比对到所述参考序列的相同染色体的正链。
  8. 权利要求6或7的装置，其特征在于，执行所述数据处理程序还包括实现数据格式转换，所述数据格式转换包括转换所述比对结果或所述唯一比对结果的格式。
  9. 权利要求1-8任一装置，其特征在于，消除所述比对结果或者所述唯一比对结果中的每个读段的缺口包括，
    若所述读段包含正缺口，以N填补所述正缺口的大小，
    若所述读段包含负缺口，去除所述负缺口，其中，
    N为A、T、C或G。
  10. 权利要求1-9任一装置，其特征在于，所述通用比对结果的格式为SAM或BAM。
  11. 一种测序数据处理系统，其包括一主机和一显示装置，其特征在于，所述系统还包括权利要求1-10任一所述的测序数据处理装置。
  12. 一种测序数据处理方法，其特征在于，包括如下步骤，
    获取测序数据，所述测序数据包括多对读段对，每对读段对由两个读段组成，分别来自一条染色体片段的两个位置，每对读长对中的两个读段分别来自所述染色体片段的正链和负链，或者每对读长对中的两个读段都来自所述染色体片段的正链或所述染色体片段的负链，每个读段都包含缺口，将一对读段对的两个读段分别定义为左臂和右臂；
    将所述测序数据与参考序列比对，获得比对结果，所述比对结果包括多个所述读段对的比对结果，和/或，
    所述比对结果包括多个所述左臂的比对结果和多个所述右臂的比对结果；
    消除所述比对结果中的每个读段的缺口，获得通用比对结果。
  13. 权利要求12的方法，其特征在于，获取所述测序数据包括构建测序文库，获得测序文库，所述测序文库为单链环状DNA文库，所述测序文库由所述染色体片段的一条链和至少一个预定DNA序列构成。
  14. 权利要求12的方法，其特征在于，所述每对读段分别来自所述染色体片段的两端。
  15. 权利要求14的方法，其特征在于，所述获取测序结果包括测序文库构建，获得测序文库，所述测序文库为单链环状DNA文库，所述测序文库由所述染色体片段的一条链和连接所述一条链的两端的一个预定DNA序列构成。
  16. 权利要求15的方法，其特征在于，构建所述测序文库包括，
    (1)提取待测核酸；
    (2)末端磷酸化所述核酸，获得末端磷酸化产物；
    (3)末端修复所述末端磷酸化产物，获得末端修复产物；
    (4)将第一序列和第二序列连接至所述末端修复产物的两端，获得第一连接产物；
    (5)利用第三序列对所述连接产物进行缺刻平移和扩增，获得扩增产物，所述第三序列为一对引物对，所述引物对的至少一条引物带有生物素标记；
    (6)利用所述生物素标记对所述扩增产物进行单链分离，获得单链产物；
    (7)利用第四序列环化所述单链产物，获得所述测序文库；其中，
    所述第四序列能够连接所述第一序列的一端和所述第二序列的一端，所述第一序列和/或所述第二序列的另一端为双脱氧核苷酸。
  17. 权利要求15的方法，其特征在于，构建所述测序文库包括，
    (1)提取待测核酸；
    (2)末端修复所述核酸，获得末端修复产物；
    (3)末端磷酸化所述末端修复产物，获得末端磷酸化产物；
    (4)将第一序列和第二序列连接至所述末端磷酸化产物的两端，获得第一连接产物；
    (5)利用第三序列对所述连接产物进行缺刻平移和扩增，获得扩增产物，所述第三序列为一对引物对，所述引物对的至少一条引物带有生物素标记；
    (6)利用所述生物素标记对所述扩增产物进行单链分离，获得单链产物；
    (7)利用第四序列环化所述单链产物，获得所述测序文库；其中，
    所述第四序列能够连接所述第一序列的一端和所述第二序列的一端，所述第一序列和/或所述第二序列的另一端为双脱氧核苷酸。
  18. 权利要求12-17任一的方法，其特征在于，所述比对包括，
    将每对读段对的左臂和右臂分别与所述参考序列比对，获得一级左比对结果和一级右比对结果，
    分别以所述一级左比对结果和所述一级右比对结果的其中一个为参考，对另一个作比对，获得二级左比对结果和二级右比对结果，
    基于所述二级左比对结果和所述二级右比对结果，获得多个所述读段对的比对结果，或者获得多个所述左臂的比对结果和多个所述右臂的比对结果。
  19. 权利要求12-18任一方法，其特征在于，所述比对包括，设置所述缺口的大小以使每个左臂或者每个右臂与所述参考序列进行多次比对。
  20. 权利要求19的方法，其特征在于，所述每个左臂或者每个右臂与参考序列进行多次比对为，将所述每个左臂或者所述每个右臂的缺口分别设置为-3nt、-2nt、-1nt、0nt、1nt、2nt、3nt、4nt、5nt、6nt和7nt，获得对应的多个读段，分别将所述对应的多个读段与所述参考序列比对。
  21. 权利要求12-20任一方法，其特征在于，所述比对结果的格式为TeraMap。
  22. 权利要求12-21任一方法，其特征在于，在所述消除比对结果中的每个读段的缺口之前，提取所述比对结果中的唯一比对结果以替换所述比对结果，所述唯一比对结果包括唯一比对上所述参考序列的多个读段对，并且每一所述读段对比对到所述参考序列的相同染色体，每一所述读段对中的两个读段的距离符合所述染色体片段的大小。
  23. 权利要求22的方法，其特征在于，对所述唯一比对结果进行修正，以使所述唯一比对结果中的每一对读段对比对到所述参考序列的相同染色体的正链。
  24. 权利要求22或23的方法，其特征在于，获得所述通用比对结果还包括，对所述比对结果或所述唯一比对结果进行数据格式转换。
  25. 权利要求12-24任一方法，其特征在于，消除所述比对结果或者所述唯一比对结果中的每个读段的缺口包括，
    若所述读段包含正缺口，以N填补所述正缺口的大小，
    若所述读段包含负缺口，去除所述负缺口，其中，
    N为A、T、C或G。
  26. 权利要求12-25任一方法，其特征在于，所述通用比对结果的格式为SAM或BAM。
  27. 一种计算机可读存储介质，其特征在于，用于存储供计算机执行的程序，所述程序的执行包括完成权利要求12-26任一方法。
  28. 一种检测SNP的方法，其特征在于，包括，
    A.获取待测样本的核酸；
    B.对所述核酸的至少一部分进行序列测定，获得测序数据；
    C.对所述测序数据进行处理，获得通用比对结果；
    D.基于所述通用比对结果检测所述SNP；其中，
    步骤C利用权利要求1-10任一测序数据处理装置进行。
  29. 权利要求28的方法，其特征在于，B步骤包括，对所述核酸的至少一部分进行测序文库构建，获得测序文库，所述测序文库为单链环状DNA文库。
  30. 权利要求29的方法，其特征在于，所述测序文库构建包括，
    末端磷酸化所述核酸，获得末端磷酸化产物；
    末端修复所述末端磷酸化产物，获得末端修复产物；
    将第一序列和第二序列连接至所述末端修复产物的两端，获得第一连接产物；
    利用第三序列对所述连接产物进行缺刻平移和扩增，获得扩增产物，所述第三序列为一对引物对，所述引物对的至少一条引物带有生物素标记；
    利用所述生物素标记对所述扩增产物进行单链分离，获得单链产物；
    利用第四序列环化所述单链产物，获得所述测序文库，其中，
    所述第四序列能够连接所述第一序列的一端和所述第二序列的一端，所述第一序列和/或所述第二序列的另一端为双脱氧核苷酸。
  31. 权利要求29的方法，其特征在于，所述测序文库构建包括，
    末端修复所述核酸，获得末端修复产物；
    末端磷酸化所述末端修复产物，获得末端磷酸化产物；
    将第一序列和第二序列连接至所述末端磷酸化产物的两端，获得第一连接产物；
    利用第三序列对所述连接产物进行缺刻平移和扩增，获得扩增产物，所述第三序列为一对引物对，所述引物对的至少一条引物带有生物素标记；
    利用所述生物素标记对所述扩增产物进行单链分离，获得单链产物；
    利用第四序列环化所述单链产物，获得所述测序文库，其中，
    所述第四序列能够连接所述第一序列的一端和所述第二序列的一端，所述第一序列和/或所述第二序列的另一端为双脱氧核苷酸。
  32. 权利要求28-31任一方法，其特征在于，所述测序是利用组合探针锚定连接测序技术进行的。
  33. 一种SNP检测设备，其特征在于，包括，
    核酸获取装置，用以获取待测样本的核酸；
    测序装置，用以对来自所述核酸获取单元的核酸的至少一部分进行测序，获得测序数据，所述测序数据包括多对读段对，每对读段对由两个读段组成，分别来自一条染色体片段的两个位置，每对读长对中的两个读段分别来自所述染色体片段的正链和负链，或者每对读长对中的两个读段都来自所述染色体片段的正链或所述染色体的负链，每个读段都包含缺口，将一对读段对的两个读段分别定义为左臂和右臂；
    数据处理装置，用于对来自所述测序装置的测序数据进行处理，以获得通用比对结果；
    检测装置，用于基于来自所述数据处理装置的通用比对结果检测所述SNP；其中，
    所述数据处理装置包括，
    数据接收单元，用于接收来自所述测序装置的测序数据，
    处理器，用于执行数据处理程序，执行所述数据处理程序包括实现将来自数据接收单元的测序数据与参考序列作比对，获得比对结果，以及消除所述比对结果中的每个读段的缺口，获得通用比对结果，所述比对结果包括多个所述读段对的比对结果，和/或，所述比对结果包括多个所述左臂的比对结果和多个所述右臂的比对结果，以及
    至少一个存储单元，用于存储数据，其中包括所述数据处理程序。
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