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WO2018026576A1 - Analyse génomique du sang ombilical - Google Patents

Analyse génomique du sang ombilical Download PDF

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Publication number
WO2018026576A1
WO2018026576A1 PCT/US2017/043745 US2017043745W WO2018026576A1 WO 2018026576 A1 WO2018026576 A1 WO 2018026576A1 US 2017043745 W US2017043745 W US 2017043745W WO 2018026576 A1 WO2018026576 A1 WO 2018026576A1
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Prior art keywords
genetic
analysis
information
individual
cord blood
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Gregory Porreca
Stanley Lapidus
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Good Start Genetics Inc
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Good Start Genetics Inc
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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
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • 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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the invention relates to the genomic analysis of cord blood over time as new information with respect to genetic markers become available.
  • cord blood has become increasingly popular as research has shown that this cord blood can be used to help treat life-threatening diseases.
  • cord blood has been shown to be useful in the treatment of diseases and disorders, such as cancers, blood disorders, vascular disease, neurological disease or disorders, autoimmune diseases or disorders and diseases or disorders involving inflammation.
  • diseases and disorders such as cancers, blood disorders, vascular disease, neurological disease or disorders, autoimmune diseases or disorders and diseases or disorders involving inflammation.
  • cord blood can also be useful in providing genetic information on the individual, which can in turn inform treatment decisions.
  • cord blood is retrieved from the umbilical cord after the birth of a newborn for both analysis and isolation of stem cells for future use.
  • Cord blood can be retrieved from the umbilical cord in a number of ways and can then be apportioned for analysis purposes and isolation of stem cells. While analysis of cord blood around the time that it is retrieved from the placenta is helpful in providing key genetic information about the individual, new genetic markers are constantly being discovered over time. Thus, the genetic information obtained from cord blood around the time of birth will not be reflective of new discoveries with respect to genetic markers and associations between genetic markers and diseases or disorders with the passage of time.
  • cord blood is initially obtained from the umbilical cord at birth. A portion of the cord blood is used for initial genetic analysis, while the remaining portion can be placed in storage for later use. Additional samples can also be obtained from the individual, such as blood and tissue samples, upon which genetic analysis can be performed. Information obtained from the initial genetic analysis can be used to identify the risk of developing a medical condition, or whether that condition already exists, which can in turn inform treatment decisions.
  • the initial collection of one or more samples eliminates the need to collect additional samples of DNA from the individual in order to assess genetic disease risk timely.
  • the cord blood that is placed in storage can later be retrieved and utilized as new markers and treatments become available.
  • information obtained from subsequent analyses of stored cord blood is useful to identify risk associated with newly-discovered disease markers, a change in an initial risk profile based upon new information, such as new markers, subsequent phenotypic development, lifestyle and other factors that might influence the etiology of disease.
  • Individual genomic information can thus be updated over time without the need to repeatedly retrieve a sample from the patient.
  • Methods and systems of the present invention can be implemented in part through the use of one or more databases or interpretation algorithms for, inter alia, storing, processing, and delivering information.
  • the present disclosure provides methods for genetic analysis that include the steps of obtaining cord blood from an individual, conducting an analysis on a nucleic acid sample from the cord blood to determine the presence of genetic markers, providing a report identifying whether one or more genetic markers are present in the individual, and storing a portion of the cord blood.
  • the method further contemplates the receipt and analysis of new information regarding genetic markers.
  • Subsequent analyses on a sample of the cord blood can be conducted to determine the presence of newly-discovered markers.
  • a report is generated thereafter identifying whether the genetic markers are present.
  • This disclosure also provides methods for delivering personalized medical information or news/educational content that is informed by the genetics of the subject both at the time of initial analysis and over the course of time. Methods of the invention are amenable to automation.
  • the invention provides algorithms comprising a computer having a processor and accessible memory coupled to the processor.
  • a database of genetic information such as markers for disease, genetic variation, epigenetic information and the like, is stored in memory.
  • Data derived from a cord blood sample is inputted and stored.
  • the computer can then access inputted data and run one or more processes that analyze the data against the stored database, thereby to produce one or more reports that indicate a correlation, a diagnosis, a match or mismatch, or other report as desired by the operator.
  • the database comprises genomic markers associated with one or more physiological condition or function and is updated periodically through user input or through automated coupling with an instrument, such as a sequencer.
  • Input data are derived from a cord blood sample as set forth herein.
  • the computer runs one or more algorithms that produce output concerning a patient's input data against the database of stored genomic information.
  • the algorithm may be a simple matching algorithm or may be a more complex algorithm that generates correlative information across multiple genomic sites in order to produce output data indicative of a physiological condition.
  • Either or both of input data and the database of stored genomic information may be in the form of a genomic sequence, metadata, epigenomic information, copy number, single nucleotide polymorphisms, rearrangements, deletions, polygenic correlations, expression data or any other type of data created by user input.
  • cord blood data are obtained through various means of sample preparation and processing, sequence analysis, and input into a computing system according to rules specific to the relevant programming environment.
  • methods of the invention involve the use of molecular inversion probes (MIPs) to conduct genomic analysis. Once nucleic acid has been isolated or extracted from the sample, it can be subjected to capture reactions using MIPS. MIPs used in in accordance with the invention can contain a common backbone sequence and two
  • sequence reads can be mapped, using a computer system having a processor coupled to a tangible memory subsystem, the sequence reads to a reference to detect genetic information.
  • the genetic information obtained can then be associated with information on genetic markers retrieved from a database, the database configured to receive updates. From there, the genotype of the individual can be determined. Alternatively or additionally, the individual can be screened for one or more genetic diseases.
  • methods of the present disclosure contemplate treating a condition based on information obtained from the analyses.
  • Treatment can involve, for example, delivering a predetermined amount of cord blood to the individual.
  • Treatment can also involve the generation of stem cells from the cord blood and subsequent delivery of the stem cells to the individual.
  • Treatment can also involve the monitoring and potential subsequent intervention with respect to genetic conditions that are likely to occur in the subject, as identified by the analysis performed.
  • Treatment can also involve the selection of therapy based in part on results of genetic analysis, e.g. drug selection aided by pharmacogenomic information.
  • the present disclosure provides methods for genetic analysis of a sample over time, including the steps of obtaining sequence data, for example, by sequencing the nucleic acid to obtain a plurality of sequence reads, from the cord blood of an individual.
  • the sequence data can be input into a computer system having a processor coupled to a tangible memory subsystem and then associated with clinical information retrieved from a database, the database configured to receive updates to the clinical information.
  • This can entail the mapping of sequence reads to a genomic reference to identify at least one variant and storing the at least one variant in the memory subsystem as a variant call prior to associating the sequencing data with the clinical information retrieved from the database.
  • the method further contemplates providing genomic information of the individual based on the results of the associating step, receiving an update to the clinical information in the database, the update adding a genetic marker to the database, determining if the individual has the genetic biomarker, and providing updated genomic information of the individual based on the determining step.
  • the determining step can involve the association of sequencing data with updated clinical information retrieved from the database.
  • the determining step can involve obtaining additional sequencing data from the cord blood and associating this additional sequencing data with the updated clinical information retrieved from the database.
  • FIG. 1 diagrams a method of the invention according to one embodiment of the present disclosure.
  • FIG. 2 illustrates use of MIPs to capture regions of target genomic material.
  • FIG. 3 illustrates the formation and detection of a "cross -probe", or inter-probe, product.
  • FIG. 4 gives a diagram of a sequencing workflow.
  • FIG. 5 diagrams a method of the invention according to another embodiment of the present disclosure.
  • FIG. 6 illustrates a platform architecture for implementing methods of the invention.
  • FIG. 7 gives a diagram of a system of the invention.
  • FIG. 8 diagrams a workflow for clinical information.
  • FIG. 9 illustrates the notification determination process.
  • FIG. 10 is a flowchart for determining a significance of an update.
  • the present disclosure provides methods and systems useful for analyzing genetic information from cord blood over time as new information with respect to genetic markers become available.
  • the use of cord blood samples for genetic analysis alleviates the need to retrieve a sample from an individual using any number of invasive methods one or more times over a course of time.
  • the information gleaned from these genetic analyses can be then used to inform medical professionals about the risk of developing a condition, or that a condition already exists for a certain individual, which can in turn inform treatment decisions.
  • the presently disclosed methods involve the general steps of obtaining cord blood from an individual 105, conducting a first analysis on a first nucleic acid sample from the cord blood to determine the presence at least a first genetic marker 109, providing a report identifying whether the first genetic marker is present in the individual 113, later receiving updated information with respect to a second genetic marker 117, conducting a second analysis on a second nucleic acid sample to determine the presence of the second genetic marker 121, and providing a report identifying whether the second genetic marker is present in the individual 125.
  • analyses are performed at least several months after the initial or previous analyses, such as at least three, four, five, six seven, eight, nine, ten, eleven or twelve months. In other embodiments, analyses are performed at least a few years after the initial or previous analysis, such as at least two, three, four, five, six, seven, eight, nine, ten, or any other number of years. It is also to be understood that each analysis can include determining the presence of any number of genetic markers, with each subsequent analysis including determining the presence of at least one new genetic marker after new information becomes available.
  • the portion of the cord blood that is not used for an analysis can be stored in between analyses.
  • the cord blood can be stored in a blood bank, such as the Cord Blood Registry, Viacord, Family Cord, lifebankUSA, and Cord BloodBanking. Storage involves the cryopreservation of cord blood. Cryopreservation of cord blood can be done using any known means for the cryopreservation of blood. See e.g., Reboredo et al., "Collection, processing and cryopreservation of umbilical cord blood for unrelated transplantation," Bone Marrow Transplantation. 26(12): 1263-1270 (Dec 2000), incorporated by reference. The following general components are involved in the cryopreservation process, up to
  • transplant/delivery of the blood or cells Harvesting of donor cells (including the collection of the cord blood), addition of cryopreservatives, the actual freezing procedure, assessment of the viability of the frozen unit after about 72 hours, a thawing procedure at a later date, the washing and conditioning of the donor unit prior to transplant/delivery. See, Berz et al.,
  • Exemplary genetic markers include genes (e.g. any region of DNA encoding a functional product), genetic regions (e.g. regions including genes and intergenic regions), and gene products (e.g., RNA, protein, and expression of genes).
  • a genetic marker can include variations with the genes and genetic regions. Exemplary variations include, but are not limited to, a single nucleotide polymorphism, a deletion, an insertion, an inversion, a genetic rearrangement, a copy number variation, or a combination thereof.
  • Genetic markers can also include abnormal gene expression.
  • a differentially or abnormally expressed gene refers to a gene whose expression is activated to a higher or lower level in a subject suffering from a disorder relative to its expression in a normal or control subject.
  • cord blood can be retrieved from the umbilical cord by a number of techniques, such as, for example and not limitation, flowing the blood from the cord into open test tubes, using one or more syringes to aspirate the cord blood, using an
  • Cord blood is specific to an individual and thus contains the individual's nucleic acid.
  • a sample may be obtained from a tissue or body fluid that is obtained in any clinically acceptable manner.
  • Body fluids may include mucous, blood, plasma, serum, serum derivatives, bile, blood, maternal blood, phlegm, saliva, sweat, amniotic fluid, menstrual fluid, mammary fluid, follicular fluid of the ovary, fallopian tube fluid, peritoneal fluid, urine, and cerebrospinal fluid (CSF), such as lumbar or ventricular CSF.
  • a sample may also be a fine needle aspirate or biopsied tissue.
  • a sample also may be media containing cells or biological material. Samples may also be obtained from the environment (e.g., air, agricultural, water and soil) or may include research samples (e.g., products of a nucleic acid amplification reaction, or purified genomic DNA, RNA, proteins, etc.).
  • isolation, extraction or derivation of genomic nucleic acids may be performed by methods known in the art.
  • Isolating nucleic acid from a biological sample generally includes treating a biological sample in such a manner that genomic nucleic acids present in the sample are extracted and made available for analysis.
  • nucleic acids are extracted using techniques such as those described in Green & Sambrook, 2012, Molecular Cloning: A Laboratory Manual 4 edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2028 pages), the contents of which are incorporated by reference herein.
  • a kit may be used to extract DNA from tissues and bodily fluids and certain such kits are commercially available from, for example, BD Biosciences Clontech (Palo Alto, CA), Epicentre Technologies (Madison, WI), Gentra Systems, Inc. (Minneapolis, MN), and Qiagen Inc. (Valencia, CA). User guides that describe protocols are usually included in such kits.
  • Cellular extracts can be subjected to other steps to drive nucleic acid isolation toward completion by, e.g., differential precipitation, column chromatography, extraction with organic solvents, filtration,
  • genomic nucleic acid may be any sequence thereof.
  • genomic nucleic acid can be re-suspended in Qiagen DNA hydration solution, or other Tris-based buffer of a pH of around 7.5.
  • cell-free nucleic acid which are fragments of DNA or RNA present in the blood stream of an individual.
  • cell-free DNA can be obtained from the plasma or serum of an individual.
  • Cell-free nucleic acid may be isolated according to techniques known in the art and include, for example, the QIAmp system from Qiagen (Venlo, Netherlands), the Triton/Heat/Phenol protocol (THP) (Xue, et al., Optimizing the Yield and Utility of Circulating Cell-Free DNA from Plasma and Serum", Clin. Chim.
  • BL-WGA blunt-end ligation-mediated whole genome amplification
  • BL-WGA blunt-end ligation-mediated whole genome amplification
  • BL-WGA blunt-end ligation-mediated whole genome amplification
  • NucleoSpin system from Macherey-Nagel, GmbH & Co. KG (Duren, Germany).
  • a blood sample is obtained from the patient and the plasma is isolated by centrifugation.
  • the circulating cell-free nucleic acid may then be isolated by any of the techniques above.
  • nucleic acids suitable for use in aspects of the invention may include without limit genomic DNA, genomic RNA, synthesized nucleic acids, whole or partial genome amplification product, and high molecular weight nucleic acids, e.g. individual chromosomes.
  • a sample is obtained that includes double- stranded DNA, such as bulk genomic DNA from an individual, and the double-stranded DNA is then denatured.
  • double stranded nucleic acid may be denatured using any suitable method such as, for example, through the use of heat, detergent incubation, or an acidic or basic solution.
  • nucleic acids including genomic nucleic acids, can be fragmented using any of a variety of methods, such as mechanical fragmenting, chemical fragmenting, and enzymatic fragmenting. Methods of nucleic acid fragmentation are known in the art and include, but are not limited to, DNase digestion, sonication, mechanical shearing, and the like.
  • U.S. Pub 2005/0112590 provides a general overview of various methods of
  • Genomic nucleic acids can be fragmented into uniform fragments or randomly fragmented.
  • nucleic acids are fragmented to form fragments having a fragment length of about 5 kilobases or 100 kilobases. Desired fragment length and ranges of fragment lengths can be adjusted depending on the type of nucleic acid targets one seeks to capture and the design and type of probes such as molecular inversion probes (MIPs) that will be used.
  • Chemical fragmentation of genomic nucleic acids can be achieved using methods such as a hydrolysis reaction or by altering temperature or pH.
  • Nucleic acid may be fragmented by heating a nucleic acid immersed in a buffer system at a certain temperature for a certain period to time to initiate hydrolysis and thus fragment the nucleic acid.
  • the pH of the buffer system, duration of heating, and temperature can be varied to achieve a desired fragmentation of the nucleic acid.
  • Mechanical shearing of nucleic acids into fragments can be used e.g., by hydro-shearing, trituration through a needle, and sonication.
  • the nucleic acid can also be sheared via
  • Nucleic acid may also be fragmented enzymatically. Enzymatic fragmenting, also known as enzymatic cleavage, cuts nucleic acids into fragments using enzymes, such as endonucleases, exonucleases, ribozymes, and DNAzymes. Varying enzymatic fragmenting techniques are well-known in the art. Additionally, DNA may be denatured again as needed after the digestion and any other sample prep steps. In certain embodiments, the sample nucleic acid is captured or targeted using any suitable capture method or assay such as amplification with PCR, hybridization capture, or capture by probes such as MIPs.
  • any suitable capture method or assay such as amplification with PCR, hybridization capture, or capture by probes such as MIPs.
  • Amplification refers to production of additional copies of a nucleic acid sequence and is generally carried out using polymerase chain reaction (PCR) or other technologies known in the art.
  • the amplification reaction may be any amplification reaction known in the art that amplifies nucleic acid molecules such as PCR.
  • Other amplification reactions include nested PCR, PCR- single strand conformation polymorphism, ligase chain reaction, strand displacement
  • amplification and restriction fragments length polymorphism transcription based amplification system, rolling circle amplification, and hyper-branched rolling circle amplification, quantitative PCR, quantitative fluorescent PCR (QF-PCR), multiplex fluorescent PCR (MF-PCR), real time PCR (RTPCR), restriction fragment length polymorphism PCR (PCR-RFLP), in situ rolling circle amplification (RCA), bridge PCR, picotiter PCR, emulsion PCR, transcription
  • QF-PCR quantitative fluorescent PCR
  • MF-PCR multiplex fluorescent PCR
  • RTPCR real time PCR
  • PCR-RFLP restriction fragment length polymorphism PCR
  • RCA in situ rolling circle amplification
  • bridge PCR picotiter PCR
  • emulsion PCR transcription
  • nucleic acid based sequence amplification amplification methods that can be used include those described in U.S. Pats. 5,242,794; 5,494,810; 4,988,617; and 6,582,938.
  • the amplification reaction is PCR as described, for example, U.S. Pat. 4,683,195; and U.S. Pat. 4,683,202, hereby incorporated by reference.
  • Primers for PCR, sequencing, and other methods can be prepared by cloning, direct chemical synthesis, and other methods known in the art. Primers can also be obtained from commercial sources such as Eurofins MWG Operon (Huntsville, AL) or Life Technologies (Carlsbad, CA).
  • Hybrid capture probes using selectable oligonucleotides can also be used to obtain nucleic acid of interest. See for example, Lapidus (U.S. patent number 7,666,593), the content of which is incorporated by reference herein in its entirety.
  • Conventional methods for making and using hybridization probes can be found in standard laboratory manuals such as: Genome Analysis: A Laboratory Manual Series (Vols. I-IV), Cold Spring Harbor Laboratory Press; PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratory Press; and Sambrook, J et al., (2001) Molecular Cloning: A Laboratory Manual, 2nd ed. (Vols. 1-3), Cold Spring Harbor Laboratory Press.
  • FIG. 2 illustrates use of MIPs 201 to capture regions of target genomic material 203, such as one or more genetic markers, for amplification and sequencing.
  • Each MIP 201 contains a common backbone sequence and two complementary arms that are annealed to a DNA sample of interest.
  • a polymerase 205 is utilized to fill in the gap between each of the two arms, and a ligase 221 is then utilized to create a set of circular molecules. Capture efficiency of the MIP to the target sequence on the nucleic acid fragment can be optimized by lengthening the hybridization and gap-filing incubation periods.
  • the resultant circular molecules 211 can be amplified using polymerase chain reaction to generate a targeted sequencing library.
  • MIPs can be used to detect or amplify particular nucleic acid sequences in complex mixtures.
  • Use of molecular inversion probes has been demonstrated for detection of single nucleotide polymorphisms (Hardenbol et al., 2005, Highly multiplexed molecular inversion probe genotyping: over 10,000 targeted SNPs genotyped in a single tube assay, Genome Res 15:269-75) and for preparative amplification of large sets of exons (Porreca et al., 2007,
  • MIPs can also provide a multiplexed breakpoint detection and localization assay that can include identifying and reporting mutations or variants such as substitutions or small indels.
  • the breakpoints detected in the multiplex assays may be associated with chromosomal rearrangements such as translocations, inversions, deletions, repetitions, wherein large segments (e.g., dozens or hundreds of base-pairs) of the genome are involved.
  • Another benefit of the disclosed probe-based methods is that the methods may operate successfully with very small amounts of original target nucleic acid, unlike array-based methods, which require an abundance of target.
  • the amount of target nucleic acid and probe used for each reaction is normalized to avoid any observed differences being caused by differences in concentrations or ratios.
  • the genomic DNA concentration is read using a standard spectrophotometer or by fluorescence (e.g., using a fluorescent intercalating dye).
  • the probe concentration may be determined experimentally or using information specified by the probe manufacturer.
  • a genomic locus Once a genomic locus has been captured, it may be amplified and/or sequenced in a reaction involving one or more primers.
  • the amount of primer added for each reaction can range from 0.1 pmol to 1 nmol, 0.15 pmol to 1.5 nmol (for example around 1.5 pmol). However, other amounts (e.g., lower, higher, or intermediate amounts) may be used.
  • a targeting arm may be designed to hybridize (e.g., be complementary) to either strand of a genetic locus of interest if the nucleic acid being analyzed is DNA (e.g., genomic DNA). For MIP probes, whichever strand is selected for one targeting arm will be used for the other one.
  • a targeting arm should be designed to hybridize to the transcribed RNA.
  • the probes should be designed to be complementary to the reverse complement of the transcribed strand.
  • MIP probes referred to herein as "capturing" a target sequence are actually capturing it by template-based synthesis rather than by capturing the actual target molecule (other than for example in the initial stage when the arms hybridize to it or in the sense that the target molecule can remain bound to the extended MIP product until it is denatured or otherwise removed).
  • a targeting arm may include a sequence that is complementary to one allele or mutation (e.g., a SNP or other polymorphism, a mutation, etc.) so that the probe will preferentially hybridize (and capture) target nucleic acids having that allele or mutation.
  • Sequence tags also referred to as barcodes
  • Uniformity and reproducibility can be increased by designing multiple probes per target, such that each base in the target is captured by more than one probe.
  • the length of a capture molecule on a nucleic acid fragment may be selected based upon multiple considerations. For example, where analysis of a target involves sequencing, e.g., with a next-generation sequencer, the target length should typically match the sequencing read-length so that shotgun library construction is not necessary.
  • captured nucleic acids may be sequenced using any suitable sequencing technique as aspects of the invention are not limited in this respect.
  • Methods of the invention also provide for combining the method of fragmenting the nucleic acid prior to capture with other MIP capture techniques that are designed to increase target uniformity, reproducibility, and specificity. Other MIP capture techniques are shown in U.S. Pub. 2012/0165202, incorporated by reference.
  • the set of probes for a given target can be designed to 'tile' across the target, capturing the target as a series of shorter sub targets.
  • some probes in the set capture flanking non-target sequence.
  • the set can be designed to 'stagger' the exact positions of the hybridization regions flanking the target, capturing the full target (and in some cases capturing flanking non-target sequence) with multiple probes having different targeting arms, obviating the need for tiling.
  • probes targeting pathological loci can be adjusted by changing the number of different MIPs used to capture a given molecule.
  • Probes for MIP capture reactions may be synthesized on programmable microarrays to provide the large number of sequences required.
  • the result of MIP capture as described in FIG. 2 includes one or more circular target probes, which then can be processed in a variety of ways.
  • Adaptors for sequencing may be attached during common linker-mediated PCR, resulting in a library with non-random, fixed starting points for sequencing.
  • a common linker-mediated PCR is performed on the circle target probes, and the post-capture amplicons are linearly concatenated, sheared, and attached to adaptors for sequencing.
  • Methods for shearing the linear concatenated captured targets can include any of the methods disclosed for fragmenting nucleic acids discussed above.
  • performing a hydrolysis reaction on the captured amplicons in the presence of heat is the desired method of shearing for library production.
  • the invention can include the formation and detection of "cross-probe", or inter-probe, product.
  • FIG. 3 illustrates the formation and detection of a "cross -probe", or inter-probe, product
  • probes normally self-ligate into circular molecules, in the case of a large deletion or other classes of structural variants, one probe may hybridize to the target by only one probe arm.
  • One probe arm from each of two probes spanning a deletion become ligated to each other. This ligation can include an extension, or "fill-in", step.
  • Cross-probe products 309 may be amplified even when exonucleases are utilized to digest linear products.
  • Inter-probe product 309 may be made resistant to exonuclease digestion via the inclusion of a phosphorothioate base or bases in the backbone of probes 201.
  • Inter-probe product 309 may be sequenced along with circularized probe molecules. Additionally or alternatively, inter-probe product may be detected in parallel to or instead of sequencing to provide for detection of a deletion or other such chromosomal abnormality. Any suitable approach can be used to detect or describe deletion 303. In some embodiments, the detection of any inter-probe product 309 provides the inference that the patient's target DNA 203 includes the deletion 303 (that is, the very act of detection means that a breakpoint or deletion is included in a report). One approach to detection of deletions or breakpoints includes sequencing the inter-probe product 309. Methods may include attachment of amplification or sequencing adaptors or barcodes or a combination thereof to target DNA captured by probes.
  • Amplification or sequencing adapters or barcodes, or a combination thereof may be attached to the fragmented nucleic acid.
  • Such molecules may be commercially obtained, such as from Integrated DNA Technologies (Coralville, IA).
  • sequences are attached to the template nucleic acid molecule with an enzyme such as a ligase.
  • Suitable ligases include T4 DNA ligase and T4 RNA ligase, available commercially from New England Biolabs (Ipswich, MA).
  • the ligation may be blunt ended or via use of complementary overhanging ends.
  • the ends of the fragments may be repaired, trimmed (e.g.
  • end repair is performed to generate blunt end 5' phosphorylated nucleic acid ends using commercial kits, such as those available from
  • the ends may be treated with a polymerase and dATP to form a template independent addition to the 3 '-end and the 5'- end of the fragments, thus producing a single A overhanging.
  • This single A can guide ligation of fragments with a single T overhanging from the 5 '-end in a method referred to as T-A cloning.
  • the ends may be left as-is, i.e., ragged ends.
  • double stranded oligonucleotides with complementary overhanging ends are used.
  • one or more barcodes is or are attached to each, any, or all of the fragments. In one embodiment, at least two barcodes are attached to each, any, or all of the fragments.
  • a barcode sequence generally includes certain features that make the sequence useful in sequencing reactions. The barcode sequences are designed such that each sequence is correlated to a particular portion of nucleic acid, allowing sequence reads to be correlated back to the portion from which they came. Methods of designing sets of barcode sequences are shown for example in U.S. Pat. 6,235,475, the contents of which are incorporated by reference herein in their entirety. In certain embodiments, the barcode sequences range from about 2 nucleotides to about 50 nucleotides.
  • the barcode sequences range from about 4 nucleotides to about 20 nucleotides.
  • the barcode sequences are attached to the template nucleic acid molecule, e.g., with an enzyme.
  • the enzyme may be a ligase or a polymerase, as discussed above. Attaching bar code sequences to nucleic acid templates is shown in U.S. Pub. 2008/0081330 and U.S. Pub. 2011/0301042, the content of each of which is incorporated by reference herein in its entirety. Methods for designing sets of bar code sequences and other methods for attaching barcode sequences are shown in U.S. Pats.
  • Bar code sequences can be incorporated via a PCR reaction as part of the PCR primer. After any processing steps (e.g., obtaining, isolating, fragmenting, amplification, or barcoding), nucleic acid can be sequenced.
  • Sequencing may be by any method known in the art.
  • DNA sequencing techniques include classic dideoxy sequencing reactions (Sanger method) using labeled terminators or primers and gel separation in slab or capillary, sequencing by synthesis using reversibly terminated labeled nucleotides, pyrosequencing, 454 sequencing, Illumina/Solexa sequencing, allele specific hybridization to a library of labeled oligonucleotide probes, sequencing by synthesis using allele specific hybridization to a library of labeled clones that is followed by ligation, real time monitoring of the incorporation of labeled nucleotides during a polymerization step, polony sequencing, and SOLiD sequencing.
  • Separated molecules may be sequenced by sequential or single extension reactions using polymerases or ligases as well as by single or sequential differential hybridizations with libraries of probes.
  • Sequencing methods in accordance with the present invention preferably high throughput sequencing methods, such as Illumina sequencing, can be used to determine the complete DNA sequence of an individual's genome, otherwise known as whole genome sequencing (WGS).
  • WGS whole genome sequencing
  • a sequencing technique that can be used includes, for example, Illumina sequencing.
  • Illumina sequencing is based on the amplification of DNA on a solid surface using fold-back PCR and anchored primers. Genomic DNA is fragmented, and adapters are added to the 5' and 3' ends of the fragments. DNA fragments that are attached to the surface of flow cell channels are extended and bridge amplified. The fragments become double stranded, and the double stranded molecules are denatured. Multiple cycles of the solid-phase amplification followed by denaturation can create several million clusters of approximately 1,000 copies of single- stranded DNA molecules of the same template in each channel of the flow cell. Primers, DNA
  • Reads generally include sequences of nucleotide data wherein read length may be associated with sequencing technology.
  • SMRT single-molecule real-time sequencing technology of Pacific Bio produces reads thousands of base-pairs in length.
  • read length may be about 700 bp in length.
  • reads are less than about 500 bases in length, or less than about 150 bases in length, or less than about 90 bases in length.
  • reads are between about 80 and about 90 bases, e.g., about 85 bases in length. In some embodiments, these are very short reads, i.e., less than about 50 or about 30 bases in length.
  • Sequence reads 251 can be analyzed, for example, to detect variants and, ultimately genotype.
  • FIG. 4 gives a diagram of a workflow for sequencing using MIPS.
  • Genomic DNA 203 is used as a starting sample and is exposed to a plurality of MIPs 201. Hybridization of the MIPs provides circularized probe product 211.
  • Barcode PCR may be performed to provide amplicon material for sequencing and to attach sample- specific molecular barcodes. The amplicons and barcodes may then be sequenced. Sequencing produces a plurality of sequence reads that may be analyzed for variants.
  • Sequence read data can be stored in any suitable file format including, for example, VCF files, FASTA files or FASTQ files, as are known to those of skill in the art.
  • PCR product is pooled and sequenced (e.g., on an Illumina HiSeq 2000).
  • Raw .bcl files are converted to qseq files using bclConverter (Illumina).
  • FASTQ files are generated by "de-barcoding" genomic reads using the associated barcode reads; reads for which barcodes yield no exact match to an expected barcode, or contain one or more low-quality base calls, may be discarded.
  • Reads may be stored in any suitable format such as, for example, FASTA or FASTQ format.
  • FASTA is originally a computer program for searching sequence databases and the name FASTA has come to also refer to a standard file format. See Pearson & Lipman, 1988, Improved tools for biological sequence comparison, PNAS 85:2444-2448.
  • a sequence in FASTA format begins with a single-line description, followed by lines of sequence data. The description line is distinguished from the sequence data by a greater-than (">") symbol in the first column. The word following the ">" symbol is the identifier of the sequence, and the rest of the line is the description (both are optional). There should be no space between the ">" and the first letter of the identifier. It is recommended that all lines of text be shorter than 80 characters. The sequence ends if another line starting with a ">" appears; this indicates the start of another sequence.
  • the FASTQ format is a text-based format for storing both a biological sequence (usually nucleotide sequence) and its corresponding quality scores. It is similar to the FASTA format but with quality scores following the sequence data. Both the sequence letter and quality score are encoded with a single ASCII character for brevity.
  • the FASTQ format is a de facto standard for storing the output of high throughput sequencing instruments such as the Illumina Genome Analyzer. Cock et al., 2009, The Sanger FASTQ file format for sequences with quality scores, and the Solexa/Illumina FASTQ variants, Nucleic Acids Res 38(6): 1767- 1771.
  • meta information includes the description line and not the lines of sequence data.
  • the meta information includes the quality scores.
  • the sequence data begins after the description line and is present typically using some subset of IUPAC ambiguity codes optionally with In a preferred embodiment, the sequence data will use the A, T, C, G, and N characters, optionally including "-" or U as-needed (e.g., to represent gaps or uracil, respectively).
  • reads may be mapped to a reference using assembly and alignment techniques known in the art or developed for use in the workflow.
  • Various strategies for the alignment and assembly of sequence reads including the assembly of sequence reads into contigs, are described in detail in U.S. Pat. 8,209,130, incorporated herein by reference.
  • Strategies may include (i) assembling reads into contigs and aligning the contigs to a reference; (ii) aligning individual reads to the reference; (iii) assembling reads into contigs, aligning the contigs to a reference, and aligning the individual reads to the contigs; or (iv) other strategies known to be developed or known in the art.
  • Sequence assembly can be done by methods known in the art including reference-based assemblies, de novo assemblies, assembly by alignment, or combination methods. Sequence assembly is described in U.S. Pat. 8,165,821; U.S. Pat.
  • Sequence assembly or mapping may employ assembly steps, alignment steps, or both. Assembly can be implemented, for example, by the program 'The Short Sequence Assembly by k-mer search and 3' read Extension ' (SSAKE), from Canada's Michael Smith Genome Sciences Centre
  • SSAKE cycles through a table of reads and searches a prefix tree for the longest possible overlap between any two sequences.
  • SSAKE clusters reads into contigs.
  • One read assembly program is Forge Genome Assembler, written by Spotify Piatt and
  • Forge distributes its computational and memory consumption to multiple nodes, if available, and has therefore the potential to assemble large sets of reads. Forge was written in C++ using the parallel MPI library. Forge can handle mixtures of reads, e.g., Sanger, 454, and Illumina reads.
  • Velvet Another exemplary read assembly program known in the art is Velvet, available through the web site of the European Bioinformatics Institute (Hinxton, UK) (Zerbino & Birney, Velvet: Algorithms for de novo short read assembly using de Bruijn graphs, Genome Research 18(5):821-829). Velvet implements an approach based on de Bruijn graphs, uses information from read pairs, and implements various error correction steps.
  • Read assembly can be performed with the programs from the package SOAP, available through the website of Beijing Genomics Institute (Beijing, CN) or BGI Americas Corporation (Cambridge, MA).
  • SOAPdenovo program implements a de Bruijn graph approach.
  • SOAP3/GPU aligns short reads to a reference sequence.
  • ABySS Another read assembly program is ABySS, from Canada's Michael Smith Genome Sciences Centre (Vancouver, B.C., CA) (Simpson et al., 2009, ABySS: A parallel assembler for short read sequence data, Genome Res., 19(6): 1117-23). ABySS uses the de Bruijn graph approach and runs in a parallel environment.
  • Read assembly can also be done by Roche's GS De Novo Assembler, known as gsAssembler or Newbler (NEW assemBLER), which is designed to assemble reads from the Roche 454 sequencer (described, e.g., in Kumar & Blaxter, 2010, Comparing de novo assemblers for 454 transcriptome data, Genomics 11:571 and Margulies 2005).
  • Newbler accepts 454 Fix Standard reads and 454 Titanium reads as well as single and paired-end reads and optionally Sanger reads. Newbler is run on Linux, in either 32 bit or 64 bit versions. Newbler can be accessed via a command-line or a Java-based GUI interface.
  • Assembled sequence reads may preferably be aligned to a reference. Methods for alignment and known in the art and may make use of a computer program that performs alignment, such as Burrows-Wheeler Aligner. Aligned or assembled sequence reads may be analyzed for the detection of mutations.
  • analyzing the reads includes assembling the sequence reads and then genotyping the assembled reads.
  • reads are aligned to hg 18 on a per-sample basis using Burrows- Wheeler Aligner version 0.5.7 for short alignments, and genotype calls are made using Genome Analysis Toolkit. See McKenna et al., 2010, The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data, Genome Res 20(9): 1297-1303 (aka the GATK program). High-confidence genotype calls may be defined as having depth >50 and strand bias score ⁇ 0. De-barcoded fastq files are obtained as described above and partitioned by capture region (exon) using the target arm sequence as a unique key.
  • Reads are assembled in parallel by exon using SSAKE version 3.7 with parameters "-m 30 -o 15".
  • the resulting contiguous sequences can be aligned to hgl8 (e.g., using BWA version 0.5.7 for long alignments with parameter "-r 1").
  • short-read alignment is performed as described above, except that sample contigs (rather than hgl8) are used as the input reference sequence.
  • Software may be developed in Java to accurately transfer coordinate and variant data (gaps) from local sample space to global reference space for every BAM-formatted alignment. Genotyping and base- quality recalibration may be performed on the coordinate-translated BAM files using the GATK program.
  • any or all of the steps of the invention are automated.
  • a Perl script or shell script can be written to invoke any of the various programs discussed above (see, e.g., Tisdall, Mastering Perl for Bioinformatics, O'Reilly & Associates, Inc., Sebastopol, CA 2003; Michael, R., Mastering Unix Shell Scripting, Wiley Publishing, Inc., Indianapolis, Indiana 2003).
  • methods of the invention may be embodied wholly or partially in one or more dedicated programs, for example, each optionally written in a compiled language such as C++ then compiled and distributed as a binary.
  • Methods of the invention may be implemented wholly or in part as modules within, or by invoking functionality within, existing sequence analysis platforms.
  • methods of the invention include a number of steps that are all invoked automatically responsive to a single starting queue (e.g., one or a combination of triggering events sourced from human activity, another computer program, or a machine).
  • a single starting queue e.g., one or a combination of triggering events sourced from human activity, another computer program, or a machine.
  • the invention provides methods in which any or the steps or any combination of the steps can occur automatically responsive to a queue.
  • Automatically generally means without intervening human input, influence, or interaction (i.e., responsive only to original or pre-queue human activity).
  • mapping sequence reads to a reference may produce output such as a text file or an XML file containing sequence data such as a sequence of the nucleic acid aligned to a sequence of the reference genome.
  • mapping reads to a reference produces results stored in SAM or BAM file (e.g., as shown in FIG. 4) and such results may contain coordinates or a string describing one or more mutations in the subject nucleic acid relative to the reference genome.
  • Alignment strings known in the art include Simple UnGapped Alignment Report (SUGAR), Verbose Useful Labeled Gapped Alignment Report (VULGAR), and Compact Idiosyncratic Gapped Alignment Report (CIGAR).
  • SUGAR Simple UnGapped Alignment Report
  • VULGAR Verbose Useful Labeled Gapped Alignment Report
  • CIGAR Compact Idiosyncratic Gapped Alignment Report
  • a sequence alignment is produced—such as, for example, a sequence alignment map (SAM) or binary alignment map (BAM) file— comprising a CIGAR string (the SAM format is described, e.g., in Li, et al., The Sequence Alignment/Map format and SAMtools, Bioinformatics, 2009, 25(16):2078-9).
  • SAM sequence alignment map
  • BAM binary alignment map
  • CIGAR displays or includes gapped alignments one-per-line.
  • CIGAR is a compressed pairwise alignment format reported as a CIGAR string.
  • a CIGAR string is useful for representing long (e.g. genomic) pairwise alignments.
  • a CIGAR string is used in SAM format to represent alignments of reads to a reference genome sequence.
  • the CIGAR string defines the sequence of matches/mismatches and deletions (or gaps). For example, the CIGAR string 2MD3M2D2M will mean that the alignment contains 2 matches, 1 deletion (number 1 is omitted in order to save space), 3 matches, 2 deletions and 2 matches. In general, for carrier screening or other assays, sequencing results will be used in genotyping. Output from mapping may be stored in a SAM or BAM file, in a variant call format
  • VCF VCF file
  • a typical VCF file will include a header section and a data section.
  • the header contains an arbitrary number of meta-information lines, each starting with characters '##', and a TAB delimited field definition line starting with a single '#' character.
  • the field definition line names eight mandatory columns and the body section contains lines of data populating the columns defined by the field definition line.
  • the VCF format is described in Danecek et al., 2011, The variant call format and VCFtools, Bioinformatics 27(15):2156-2158.
  • the data contained in a VCF file represents the variants, or mutations, that are found in the nucleic acid that was obtained from the sample from the patient and sequenced.
  • mutation refers to a change in genetic information and has come to refer to the present genotype that results from a mutation.
  • mutations include different types of mutations such as substitutions, insertions or deletions (INDELs), translocations, inversions, chromosomal abnormalities, and others.
  • INDELs substitutions, insertions or deletions
  • translocations inversions
  • chromosomal abnormalities and others.
  • an absolute allele frequency is not determined (i.e., not every human on the planet is genotyped) but allele frequency refers to a calculated probable allele frequency based on sampling and known statistical methods and often an allele frequency is reported in terms of a certain population such as humans of a certain ethnicity.
  • Variant can be taken to be roughly synonymous to mutation but referring to a genotype being described in comparison or with reference to a reference genotype or genome.
  • variant describes a genotype feature in comparison to a reference such as the human genome (e.g., hgl8 or hgl9 which may be taken as a wild type).
  • Methods described herein generate data representing a location of a breakpoint or deletion in the genome of a patient, which data may further also represent one or more mutations, or "variant calls.”
  • a description of a mutation may be provided according to a systematic nomenclature.
  • a variant can be described by a systematic comparison to a specified reference which is assumed to be unchanging and identified by a unique label such as a name or accession number.
  • the A of the ATG start codon is denoted nucleotide +1 and the nucleotide 5' to +1 is -1 (there is no zero).
  • a systematic name can be used to describe a number of variant types including, for example, substitutions, deletions, insertions, and variable copy numbers.
  • a substitution name starts with a number followed by a "from to" markup.
  • 199A>G shows that at position 199 of the reference sequence, A is replaced by a G.
  • a deletion is shown by "del" after the number.
  • 223delT shows the deletion of T at nt
  • 997-999del shows the deletion of three nucleotides (alternatively, this mutation can be denoted as 997-999delTTC).
  • the ⁇ nt is arbitrarily assigned; e.g.
  • a TG deletion is designated 1997-1998delTG or 1997-1998del (where 1997 is the first T before C). Insertions are shown by ins after an interval. Thus 200-201insT denotes that T was inserted between nts 200 and 201. Variable short repeats appear as 997(GT)N-N' . Here, 997 is the first nucleotide of the dinucleotide GT, which is repeated N to N' times in the population.
  • Variants in introns can use the intron number with a positive number indicating a distance from the G of the invariant donor GU or a negative number indicating a distance from an invariant G of the acceptor site AG.
  • IVS3+1C>T shows a C to T substitution at nt +1 of intron 3.
  • cDNA nucleotide numbering may be used to show the location of the mutation, for example, in an intron.
  • C.1999+1C>T denotes the C to T substitution at nt +1 after nucleotide 1997 of the cDNA.
  • c.l997-2A>C shows the A to C substitution at nt -2 upstream of nucleotide 1997 of the cDNA.
  • the mutation can also be designated by the nt number of the reference sequence.
  • a patient's genome may vary by more than one mutation, or by a complex mutation that is describable by more than one character string or systematic name.
  • the invention further provides systems and methods for describing more than one variant using a systematic name. For example, two mutations in the same allele can be listed within brackets as follows: [1997G>T; 2001A>C]. Systematic nomenclature is discussed in den Dunnen &
  • Any suitable gene may be screened using methods of the invention.
  • methods of the invention can be used to screen for disorders, such as recessive Mendelian disorders.
  • Genetic disorders and their associated genes that can be screened using methods of the invention include, for example and not limitation, Alpha-Thalassemia, Beta- Thalassemia, Fragile X Syndrome (FXS), Gaucher Disease (GD), Joubert Syndrome 2 (JBTS2), Nemaline Myopathy, Sickle Cell Disease (SCD), Spinal Muscular Atrophy (SMA), Walker- Warbug Syndrome (WWS), Canavan disease (ASPA), cystic fibrosis (CFTR), glycogen storage disorder type la (G6PC), Niemann-Pick disease (SMPD1), Tay-Sachs disease (HEXA), Bloom syndrome (BLM), Fanconi anemia C (FANCC), familial Hyperinsulinism (ABCC8), maple syrup urine disease type 1A (BCKDHA) and type IB (BCKDHB), Usher syndrome type III (CLRN1), dihydrolipoamide dehydrogenase deficiency (D
  • IKBKAP mucolipidosis type IV
  • PCDH15 Usher syndrome type IF
  • Other genetic disorders for which the presently disclosed methods can be used to screen for include cardiomyopathy, genetic hearing loss, retinoblastoma, intellectual disability/delay/autism spectrum disorder (ASD). Additionally, methods of the present disclosure can also be used to determine pharmacogenomics status of a subject.
  • Methods of the invention may include detecting and describing genetic markers such as mutations in an individual's genome.
  • a database can be used for lookup, comparisons, or storage. Once the genetic markers have been identified, a report can be provided to a user, such as a medical professional and/or the individual.
  • a database can be used, the database being configured to receive updated genetic marker information, such that subsequent analysis of an individual's nucleic acid from cord blood can be conducted.
  • a novel mutation is detected, it is classified and if pathogenic according to classification criteria, then it is entered into a database for use in future assays and comparisons.
  • a database architecture see U.S. Pat. 8,812,422, incorporated by reference.
  • single nucleotide substitutions or insertions/deletions not exceeding lObp e.g., that are located in exons or within the first lObp of an intron
  • gross chromosomal rearrangements such as deletions, translocations, and inversions may be detected or stored.
  • Variants may be named according to HGVS- recommended nomenclature or any other systematic mutation nomenclature.
  • Mutations in the database may be classified. Classification criteria described here apply to recessive Mendelian disorders and highly penetrant variants with relatively large effects. Classification criteria may follow recommendations in the literature: Richards et al., ACMG recommendations for standards for interpretation and reporting of sequence variations: Revisions 2007, Genet Med 2008, 10:294- 300; Maddalena et al., Technical standards and guidelines: molecular genetic testing for ultra- rare disorders, Genet Med 2005, 7:571-83; and Strom CM, Mutation detection, interpretation, and applications in the clinical laboratory setting, Mutat Res 2005, 573: 160-7, each incorporated by reference.
  • Classification may be based on any suitable combination of sequence-based evidence (e.g., being a truncating mutation), experimental evidence, or genetic evidence (e.g., classified as pathogenic based on genetic evidence if it was a founder variant, or if there was statistical evidence showing the variant was significantly more frequent in affected individuals than in controls; see Mac Arthur et al., Guidelines for investigating causality of sequence variants in human disease, Nature 2014, 508:469-76).
  • sequence-based evidence e.g., being a truncating mutation
  • experimental evidence e.g., being a truncating mutation
  • genetic evidence e.g., classified as pathogenic based on genetic evidence if it was a founder variant, or if there was statistical evidence showing the variant was significantly more frequent in affected individuals than in controls; see Mac Arthur et al., Guidelines for investigating causality of sequence variants in human disease, Nature 2014, 508:469-76).
  • FIG. 5 provides a method 401 according to one embodiment of the present disclosure that specifically involves the use of a database.
  • the method 401 involves obtaining sequence data from the cord blood of an individual 405, for example extracting the cord blood from the placenta after the birth of the individual.
  • the sequence data is then input into a computer system having a processor coupled to a tangible memory subsystem 409. From there, the sequence data is associated with clinical information retrieved from one or more databases, the databases configure to receive updates to the clinical information 413. Genetic information of the individual can then be provided based on the results of the association 417.
  • the method also involves the incorporation of updates to clinical information that occur once the initial association has been completed. When an update to the clinical information occurs, the update is received in one or more of the databases 421.
  • the update adds at least one genetic marker to the database.
  • a determination as to whether the individual has the genetic biomarker is then made 425. Based on this determination, updated genetic information of the individual is provided 429.
  • FIG. 6 illustrates a platform architecture for implementing methods, such as method 401, of the invention.
  • the platform may be built on a web services infrastructure such as, for example, Amazon Web Services (AWS).
  • the services infrastructure may provide storage and compute modules or functionality.
  • the raw sequence data is brought in and through assembly or variant calling is taken as the patient's genome data.
  • the scientific literature at large integrates by means of a genomic platform for biomedical analysis such as, for example, the service sold under the name GENOSPACE by Genospace (Cambridge, MA).
  • Queries against the genomic platform can provide functional information about a genetic marker, such as a variant.
  • a genetic marker such as a variant.
  • Such information may include what gene it lies within, if any; is the variant inside or outside of an intron, exon, other feature, or does it span a boundary; does the variant lie within an open reading frame; or does the variant create a frameshift or missense or nonsense mutation or premature stop codon or silent mutation.
  • Functional assessment may proceed using tools such as Genospace, Broad Inst., Signifikance, etc., assess a functional impact of a variant.
  • systems and methods of the invention may be used to retrieve medical/clinical information from an outside database.
  • the outside database can be a clinical decision support system such as UP2DATE by Wolters-Kluwer. Any suitable clinical decision support resources may be included in the outside database that is queried by the system. Other suitable resources include the medical reference resource sold under the name
  • CDS clinical decision support
  • PREDICT project uses CDS functionality of an electronic record, StarPanel, to provide active CDS.
  • PREDICT is currently designed to include both preemptive testing and "just in time,” indication-based testing.
  • Genomics Adviser is available as a stand-alone external CDS technology or it can be integrated with other applications.
  • the outside database may represent a distillation of the medical literature at large.
  • a curated database is used wherein curators work from the medical literature to keep the database up to date.
  • the outside database will include medical data and metadata, where the medical data represents the intended content (e.g., accessible by a subscriber by opening an SQL handle) and the metadata represents internal information such as a revision history.
  • an outside database is used in which each update is labeled with metadata that characterizes the update.
  • the metadata may identify the update as one or more of: correct a typo; new SNP added; clinical trial initiated; new primers published; mutation description transcluded from OMIM; author list changed.
  • a front end module provides a web- or mobile-based interface to users.
  • FIG. 7 gives a diagram of a system 501 according to embodiments of the invention.
  • System 501 may include an analysis instrument 503 which may be, for example, a sequencing instrument (e.g., a HiSeq 2500 or a MiSeq by Illumina).
  • Instrument 503 includes a data acquisition module 505 to obtain results data such as sequence read data.
  • Instrument 503 may optionally include or be operably coupled to its own, e.g., dedicated, analysis computer 533 (including an input/output mechanism, one or more processor, and memory). Additionally or alternatively, instrument 503 may be operably coupled to a server 513 or computer 549 (e.g., laptop, desktop, or tablet) via a network 509.
  • a server 513 or computer 549 e.g., laptop, desktop, or tablet
  • Computer 549 includes one or more processors and memory as well as an input/output mechanism. Where methods of the invention employ a client/server architecture, steps of methods of the invention may be performed using the server 513, which includes one or more of processors and memory, capable of obtaining data, instructions, etc., or providing results via an interface module or providing results as a file.
  • the server 513 may be provided by a single or multiple computer devices, such as the rack-mounted computers sold under the trademark BLADE by Hitachi.
  • the server 513 may be provided as a set of servers located on or off-site or both.
  • the server 513 may be owned or provided as a service.
  • the server 513 may be provided wholly or in-part as a cloud-based resources such as Amazon Web Services or Google.
  • the server 513 includes one or a plurality of local server boxes working in conjunction with a cloud resource (where local means not-cloud and includes or or off-site).
  • the server 513 may be engaged over the network 509 by the computer 549 and either or both may engage the outside database 567.
  • a computer of the invention will generally include one or more I/O device such as, for example, one or more of a video display unit (e.g., a liquid crystal display (LCD) or a cathode ray tube (CRT)), an alphanumeric input device (e.g., a keyboard), a cursor control device (e.g., a mouse), a disk drive unit, a signal generation device (e.g., a speaker), a touchscreen, an accelerometer, a microphone, a cellular radio frequency antenna, and a network interface device, which can be, for example, a network interface card (NIC), Wi-Fi card, or cellular modem.
  • a video display unit e.g., a liquid crystal display (LCD) or a cathode ray tube (CRT)
  • an alphanumeric input device e.g., a keyboard
  • a cursor control device e.g., a mouse
  • a disk drive unit e.g., a disk
  • the system 501 includes a computer 549 with a processor coupled to a tangible, non-transitory memory and one or more input/output devices.
  • the memory includes instructions that, when executed by the processor, cause the system to obtain sequence data from cord blood of an individual and analyze the sequence data for the presence of one or more known genetic markers.
  • the computer 549 may provide a report describing the presence of the markers in the individual.
  • the system is further operable to receive information that includes a new genetic marker (i.e., not yet described as a genetic marker within the system). Having received the information for the new genetic marker, the system analyzes the sequence data for the presence of the new genetic marker. The system can then provide a report that describes the presence or absence of the new genetic marker in the individual.
  • the system may obtain the information for the new genetic marker from an external system 567 such as Genospace or OMIM.
  • the system stores genetic marker information using, for example, a variant database, which may use a database structure such as that described in U.S. Pat. 8,812,422, incorporated by reference.
  • This functionally may be implemented via the use of a language such as C++ to implement a schema that includes a class for genomic feature.
  • the system can create instances of the class for any of sequence reads, variant calls, or genetic markers.
  • the abstract class includes at least a genomic position or coordinate.
  • a feature such as a sequence read, variant call, or genetic marker can be implemented by having the system create an object for the feature and store the object in the tangible, non-transitory memory.
  • the system may receive a description of the SNP and associated medial information from the external system 567.
  • the system may instantiate the class as an object in the memory.
  • the SNP is, say, c.A2650>T
  • the position or coordinate is set to 2650 and the object includes the A to T substitution.
  • the system may obtain the sequence data from the cord blood via a connection to a laboratory information management system (LIMS).
  • the LIMS may include the analysis computer 533, the sequencing instrument 503, or both.
  • the system include an Illumina HiSeq as the sequencing instrument 503.
  • the sequencing instrument sequences nucleic acid from a sample of the cord blood and the LIMS mediates transfer of the raw sequence reads to the computer system.
  • the computer system can then perform any desired steps for quality control, read assembly, and variant calling.
  • the computer system may receive the reads in FASTQ format and perform quality control by screening for reads with quality scores that meet a certain threshold.
  • the system may then perform sequence assembly and variant calling, e.g., as described in U.S. Pat. 8,209,130, incorporated by reference.
  • the system simply maps individual ones of the sequence reads to the reference to call variants and stores the variant calls in the memory.
  • the computer 549 preferably compares the sequence reads (assembled or individually) to a reference such as the human genome (e.g., hgl8).
  • the results are stored in the database as a collection of variants, relative to the human genome, for the individual. Those results may be compared to the one or more known genetic markers.
  • a feature of the system is that the database can store sequence data from multiple different cord blood samples, each from a different individual.
  • the system can analyze the database entries for the presence of the new marker and send alerts, reports, or both to the relevant clinician or individual.
  • the system is operable to receive new clinical information such as the new genetic marker and analyze the sequence data of the individual to determine the presence of the new genetic marker in the individual.
  • the system may analyze all of the sequence data in the database for multiple cord blood samples for multiple individuals. Where any of the sequence data does not include coverage that would include the new genetic marker (e.g., the new genetic marker lies within an exon not previously covered by genetic screening of cord blood), the system can initiate sequence of the cord blood samples from the cord blood bank. Thus, using the LIMS, the system can initiate retrieve of a cord blood sample from the bank. That sample can be sequenced using the sequencing instrument 503 to obtain new sequence data for the individual, in which the new sequence data can reveal the presence of the new genetic marker in the individual. And that new sequence data can be analyzed and reported using systems and methods of the invention.
  • the system may analyze all of the sequence data in the database for multiple cord blood samples for multiple individuals. Where any of the sequence data does not include coverage that would include the new genetic marker (e.g., the new genetic marker lies within an exon not previously covered by genetic screening of cord blood), the system can initiate sequence of the cord blood samples from the cord blood bank. Thus, using the LIMS, the system can initiate retrieve of
  • the system creates objects the sequence data from cord blood of an individual or the variant calls that result from comparison of the sequence data to a reference.
  • the variant calls may be stored in VCF, SAM, or BAM format as an object in the database.
  • the system stores the information for the one or more known genetic markers as one or more objects in the database.
  • the system compares the known genetic markers to the variant calls and can store the connection between the individual and the known genetic markers using index-free adjacency. That is, the object for the sequence data can be written to contain a pointer to the object(s) for the one or more known genetic markers. Using index-free adjacency, the system can rapidly scan all of the cord blood database entries for genetic markers.
  • Index-free adjacency may be implemented as described in U.S. Pub. 2016/0048608, incorporated by reference. Moreover, the system may create an object for the report describing the presence of the markers in the individual. The report may similarly be connected to the sequence data from the individual via index-free adjacency. In such implementation, when the information for the new genetic marker is received, the system may create an object for the information and link it to sequence data objects in the database, via pointers, to indicate the presence of the new genetic marker in such sequence data (where new genetic marker means not yet stored as a genetic marker in the database). When the database is implemented using index-free adjacency, upon the discovery of a new genetic marker, the system can very rapidly analyze and report all cord blood samples stored therein.
  • the system uses a computer that includes a processor coupled to a tangible, non- transitory memory and one or more input/output devices to analyze sequence data from a cord blood sample from an individual and, upon receipt of information for a new genetic marker, analyze the sequence data for the new genetic marker or generate new sequence data from an associated cord blood bank.
  • a processor coupled to a tangible, non- transitory memory and one or more input/output devices to analyze sequence data from a cord blood sample from an individual and, upon receipt of information for a new genetic marker, analyze the sequence data for the new genetic marker or generate new sequence data from an associated cord blood bank.
  • Specific implementations including objects as instances of a class of genomic feature, index-free adjacency for the analysis and reporting, and integration with an LIMS are features that may allow the system to accurately and rapidly report on the health of a large number of individuals represented in a database and associated cord blood bank.
  • FIG. 8 diagrams a workflow for the medical information.
  • the system provides a repository of patient information and meta-data, which includes clinical information, genome data, and patient subscription information.
  • the system 501 can query curated external databases 567 for disease association and reporting information.
  • variants may be associated with a disease and any additional information.
  • information may be obtained from such a source as Genospace, OMIM, or Collinso Biosciences through a systematic and semantically-controlled combination of manual and automated curation.
  • disease association provides, for a variant, any disease known to be associated with that variant.
  • the disease associations may be provided by existing internal databases.
  • functional assessment may locate a SNP within a cystic fibrosis transmembrane receptor and that SNP may already be tracked in an internal database within the server 513 (see, e.g., U.S. Pat. 8,812,422, incorporated by reference) as associated with the disease cystic fibrosis.
  • systems of the invention can include, in provided patient reports, actionable medical information.
  • the medical text can be provided by querying an outside source such as a clinical decision support resource as the outside database.
  • a clinical decision support resource as the outside database.
  • One suitable product is the clinical decision support resource offered under the trademark UP2DATE by Wolters-Kluwer.
  • Systems and methods of the invention use automated access to structured, actionable medical information for specific diseases from the outside database and provide for custom integration of updates based on new "tagged content" from the outside database.
  • Systems of the invention implement automated access to structured, actionable medical information for specific diseases. Custom integration of updates may be based on new "tagged content" from the outside database.
  • methods of the present disclosure contemplate notifying a user of updated genetic information, such as new genetic biomarkers.
  • FIG. 9 diagrams a method 701 for determining whether to notify a user of the availability of an updated report.
  • the system may provide a report for a user that includes an identity of the individual, the variant call, and the clinical information on the variant and also later provide an updated reported with the updated clinical information.
  • the initial report may be provided by querying 707 the outside database 567 for curated variant interpretation data.
  • the system 513 can determine whether an update to the clinical information has been published. This may be done by simply comparing the present information to the information that was last used to generate a report for the individual. In one embodiment, the system 513 can continue to receive, process, and store updates to clinical information without automatically notifying a user. In another embodiment, the system 513 can automatically notify the user of new clinical information, such as new genetic markers.
  • the system can evaluate 713 whether the update meets predetermined criteria for significance and notify 723 a user of updated clinical information. Alternatively, no evaluation as to significance of the update occurs before a user is notified.
  • the system may be used to compose a new report by querying 719 the outside database 567, specifically the updated data, and provide the new report, which preferably includes new clinical information associated with one or more genetic biomarkers.
  • the new clinical information associated may include one or more of functional information, a disease association, and medical information.
  • the new clinical information includes updated information about an association of a variant with a medical condition, a prognosis, a treatment regimen, or a propensity for disease.
  • FIG. 10 is a flowchart for determining a significance of an update according to one embodiment.
  • the evaluating step comprises reading metadata entered into said database.
  • the metadata may include such information as a source of the update, a date of the update, and the predetermined criteria.
  • the evaluation of the significance of the new information can take into account both the scope of the change and the impact to the particular patient.
  • the evaluation may include a scope assessment 715 and an impact assessment 721.
  • the scope assessment 715 looks at the substance of the update.
  • the curators will tag updates with metadata that characterizes the update.
  • the outside database may provide a defined schema for the metadata tags and system 513 may be
  • tags that may be read in the scope assessment, and whether the scope assessment results in a "Yes, proceed” or a "No, do not report”, may include, for example:
  • the impact assessment 721 can query whether an update has applicability to an individual with, for example, a particular mutation. For instance, where a disease phenotype is known to require an indel proximal to a SNP, for an individual with the SNP but not the indel, new medical information about the SNP may be determined to not be impactful to that individual. Thus by means of the scope assessment 715 and the impact assessment 721 some updates may be deemed trivial and ignored, for example, where a minor change is documented in incidence of a disease in some demographic. Additionally, updates need not trigger a notification if not relevant to the individual, for example, a where a SNP is linked to prostate cancer a female individual may not be given an urgent notification.
  • the update and notification steps may be performed once, multiple times, regularly, periodically, on-demand, or according to any other desired schedule (e.g., the determining, evaluating, and notifying steps are performed a plurality of times for a plurality of different updates over a period of at least a week).
  • Systems and methods of the invention provide a user interface 131 via, for example, a mobile app or a desktop web app.
  • the user interface provides personalized access to updated data. Physicians or genetic counselors or their patients may receive alerts generated by curated updates of relevant information automatically pushed out to the users. Incorporation by Reference

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Abstract

La présente invention concerne des procédés et des systèmes utiles pour analyser des informations génétiques provenant du sang ombilical et/ou d'autres échantillons d'acide nucléique au fil du temps à mesure que de nouvelles informations concernant des marqueurs génétiques deviennent disponibles. Un procédé donné à titre d'exemple comprend les étapes consistant à prélever du sang ombilical d'un individu, à effectuer une première analyse sur un premier échantillon d'acide nucléique pour déterminer la présence d'au moins un premier marqueur génétique, à fournir un rapport indiquant si le premier marqueur génétique est présent chez l'individu, à recevoir ultérieurement des informations mises à jour en ce qui concerne un deuxième marqueur génétique, à effectuer une deuxième analyse sur un deuxième échantillon d'acide nucléique afin de déterminer la présence du deuxième marqueur génétique et à fournir un rapport indiquant si le deuxième marqueur génétique est présent chez l'individu. Des informations recueillies à partir de ces analyses peuvent être utilisées par des professionnels médicaux pour informer les décisions de traitement.
PCT/US2017/043745 2016-08-04 2017-07-25 Analyse génomique du sang ombilical Ceased WO2018026576A1 (fr)

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WO2022240762A1 (fr) * 2021-05-10 2022-11-17 University Of Iowa Research Foundation Séquençage massivement parallèle ciblé destiné au dépistage de la perte auditive génétique et de la perte auditive congénitale associée au cytomégalovirus

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022240762A1 (fr) * 2021-05-10 2022-11-17 University Of Iowa Research Foundation Séquençage massivement parallèle ciblé destiné au dépistage de la perte auditive génétique et de la perte auditive congénitale associée au cytomégalovirus

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