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EP4581367A1 - Procédés de mesure de longueur de télomère - Google Patents

Procédés de mesure de longueur de télomère

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Publication number
EP4581367A1
EP4581367A1 EP23861627.0A EP23861627A EP4581367A1 EP 4581367 A1 EP4581367 A1 EP 4581367A1 EP 23861627 A EP23861627 A EP 23861627A EP 4581367 A1 EP4581367 A1 EP 4581367A1
Authority
EP
European Patent Office
Prior art keywords
telomere
sequence
tagged
dna
sequencing
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP23861627.0A
Other languages
German (de)
English (en)
Inventor
Kayarash KARIMIAN
Carol W. Greider
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Johns Hopkins University
University of California Berkeley
University of California San Diego UCSD
University of California Santa Barbara UCSB
Original Assignee
Johns Hopkins University
University of California Berkeley
University of California San Diego UCSD
University of California Santa Barbara UCSB
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Johns Hopkins University, University of California Berkeley, University of California San Diego UCSD, University of California Santa Barbara UCSB filed Critical Johns Hopkins University
Publication of EP4581367A1 publication Critical patent/EP4581367A1/fr
Pending legal-status Critical Current

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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
    • C12Q1/6811—Selection methods for production or design of target specific oligonucleotides or binding molecules
    • 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
    • C12Q2600/154—Methylation markers

Definitions

  • the present disclosure relates to the field of biotechnology, and more specifically, to measurement of mammalian telomere lengths.
  • Telomeres are the end of eukaryotic chromosomes and shorten with time. Shortened telomeres cause disease in patients with short telomere syndromes. Cancer cells must activate telomerase to maintain telomeres to overcome senescence or apoptosis due to critically short telomeres.
  • Telomere length is often measured by Southern Blot (invented in 1973). This method measures the length of all the telomeres in the cell and does not give quantitative information or information about individual telomere lengths.
  • a quantitative PCR method was developed however it has many problems with reproducibility across different labs.
  • the Single Telomere Length Analysis (STELA) method and the related Telomere Shortest Length Assay (TeSLA) targets individual telomeres, but the use of high cycles of PCR generates bias for shorter products. Additionally, the use of Southern Blotting in STELA and TeSLA makes these techniques laborious and low-throughput.
  • Telomere Flow FISH is the accepted clinical method for measuring telomere length.
  • telomere length is highly reproducible, and can report on a specific cell type, however it requires fresh blood samples and is not able to measure archival samples or tissues other than blood.
  • the method uses an average of the telomeres without the ability to report the individual lengths of telomeres. Thus, there are currently no methods that provide an accurate, high throughput, and simple approach for quantifying telomere length.
  • the present disclosure is based in part on the discovery of technology that allows for accurate high-throughput measurement of mammalian telomere lengths.
  • the methods disclosed herein include tagging of the ends of telomeres to mark their natural ends, therefore providing an accurate, high-throughput, and simple approach for quantifying telomere length.
  • telomere length of a DNA molecule from a biological sample comprising: (a) providing a telomere tagging probe comprising a biotin adapter; (b) providing a splint oligonucleotide, wherein the splint oligonucleotide specifically binds to at least a portion of a telomere on the DNA molecule and to at least a portion of the telomere tagging probe; (c) attaching the telomere tagging probe to the telomere to generate a tagged telomere sequence; (d) contacting the tagged telomere sequence with a streptavidin- functionalized bead, wherein the biotin adapter of the tagged telomere sequence binds the streptavidin-functionalized bead; (e) recovering the tagged telomere sequence; and (f) analyzing the tagged telomere sequence, thereby determining the
  • the attaching step (c) comprises ligating the telomere tagging probe to the 3’ end of the telomere.
  • the ligating comprises cycling ligation.
  • the ligating comprises a Taq DNA ligase.
  • Also provided herein are methods for determining a telomere length of a DNA molecule from a biological sample comprising: (a) providing a telomere tagging probe, wherein the telomere tagging probe comprises a unique molecular identifier (UMI) sequence; (b) providing a splint oligonucleotide, wherein the splint oligonucleotide specifically binds to at least a portion of a telomere on the DNA molecule and to at least a portion of the telomere tagging probe; (c) attaching the telomere tagging probe to the telomere to generate a tagged telomere sequence; (d) contacting the tagged telomere sequence with a forward primer and a reverse primer, wherein the forward primer binds to a subtelomere of the telomere, and the reverse primer binds to at least a portion of the telomere tagging probe; (e) amp
  • the amplification comprises PCR amplification. In some embodiments, the amplification comprises 20 rounds of PCR amplification. In some embodiments, the analyzing comprises sequencing the tagged telomere sequence. In some embodiments, the sequencing comprises long read sequencing.
  • the telomere tagging probe further comprises a sample barcode sequence that is associated with the biological sample.
  • the biological sample comprises a blood sample.
  • the biological sample comprises a tissue sample.
  • FIG. 1 shows an exemplary workflow of a tagging and sequencing method described herein as the Direct Telomere Profiling method.
  • a Telomere Tag (“TeloTag”) is biotinylated to allow enrichment of telomeres, which are then sequenced with Oxford Nanopore Technology (ONT) platforms.
  • ONT Oxford Nanopore Technology
  • FIG. 2 shows an exemplary workflow of a tagging and sequencing method described herein as the Amplified Telomere Profiling method. This method uses PCR amplification from telomeres tagged with TeloTags containing UMI barcodes followed by PacBio sequencing.
  • FIG. 3B shows a portion of a reference genome used to assess Nanopore telomere profiling to accurately measure telomere length.
  • FIG. 5A shows length bias of nanopore sequencing does not impact telomere length measured by Direct Telomere Profiling method.
  • Telomere length varies greatly between species, from approximately 300 base pairs in yeast to many kilobases in humans, and usually is composed of arrays of guanine-rich, six- to eight- base-pair-long repeats. Eukaryotic telomeres normally terminate with 3’ single-stranded-DNA overhang, which is essential for telomere maintenance and capping.
  • telomere length of a DNA molecule from a biological sample include (a) providing a telomere tagging probe comprising a biotin adapter; (b) providing a splint oligonucleotide, wherein the splint oligonucleotide specifically binds to at least a portion of a telomere on the DNA molecule and to at least a portion of the telomere tagging probe; (c) attaching the telomere tagging probe to the telomere to generate a tagged telomere sequence; (d) contacting the tagged telomere sequence with a streptavidin-functionalized bead, wherein the biotin adapter of the tagged telomere sequence binds the streptavidin-functionalized bead; (e) recovering the tagged telomere sequence; and (f) analyzing the tagged telomere sequence, thereby determining the telomere
  • Also provided herein are methods for determining a telomere length of a DNA molecule from a biological sample that include (a) providing a telomere tagging probe, wherein the telomere tagging probe comprises a unique molecular identifier (UMI) sequence; (b) providing a splint oligonucleotide, wherein the splint oligonucleotide specifically binds to at least a portion of a telomere on the DNA molecule and to at least a portion of the telomere tagging probe; (c) attaching the telomere tagging probe to the telomere to generate a tagged telomere sequence; (d) contacting the tagged telomere sequence with a forward primer and a reverse primer, wherein the forward primer binds to a subtelomere of the telomere, and the reverse primer binds to at least a portion of the telomere tagging probe; (e) amplifying the
  • methods described herein include attaching a telomere tagging probe to a 3’ end of the telomere to generate a tagged telomere sequence.
  • the attaching step comprises ligating the telomere tagging probe to the 3’ end of the telomere.
  • the ligating comprises cycling ligation.
  • the ligating comprises using a DNA ligase enzyme.
  • the DNA ligase enzyme is from a bacterium, e.g., the DNA ligase enzyme is a bacterial DNA ligase enzyme.
  • methods provided herein include use of a splint oligonucleotide comprising a nucleic acid sequence that specifically binds to at least a portion of the telomere and to at least a portion of the telomere tagging probe.
  • a “splint oligonucleotide” is an oligonucleotide that, when hybridized to other polynucleotides, acts to position the polynucleotides next to one another so that they can be attached (e.g., ligated together).
  • the splint oligonucleotide is DNA or RNA.
  • the splint oligonucleotide is between 10 and 50 oligonucleotides in length (e.g., between 10 and 45, between 10 and 40, between 10 and 35, between 10 and 30, between 10 and 25, between 10 and 20, or between 10 and 15, between 15 and 50, between 15 and 45, between 15 and 40, between 15 and 35, between 15 and 30, between 15 and 25, between 15 and 20, between 20 and 50, between 20 and 45, between 20 and 40, between 20 and 35, between 20 and 30, between 20 and 25, between 25 and 50, between 25 and 45, between 25 and 40, between 25 and 35, between 25 and 30, between 30 and 50, between 30 and 45, between 30 and 40, between 30 and 35, between 35 and 50, between 35 and 45, between 35 and 40, between 40 and 50, between 10 and 50 oligonucleotides in length (e.g., between 10 and 45, between 10 and 40, between 10 and 35, between 10 and 30, between 10 and 25, between 10 and 20, or between 10 and 15, between 15 and 50, between 15 and 45, between 15
  • the splint oligonucleotide is not blocked at its 3’ end.
  • the splint oligonucleotide comprises SEQ ID NO: 1-143.
  • the nucleic acid sequence that specifically binds to at least a portion of the telomere includes a sequence complementary to the repetitive sequence of the telomere.
  • the nucleic acid comprises a CCCTAA (SEQ ID NO: 144) sequence.
  • methods described herein include contacting the tagged telomere sequence with a streptavidin-functionalized bead, wherein the biotin adapter of the tagged telomere sequence binds with the streptavidin-functionalized bead, and recovering the tagged telomere sequence.
  • the streptavidin- functionalized bead is formulated for DNA capture.
  • the streptavidin- functionalized bead comprises MyOneTM Streptavidin Cl beads.
  • the recovering comprises using a magnet to separate the tagged telomere sequence bound to the streptavidin-functionalized bead. In some embodiments of direct telomere profiling, the recovering further comprises releasing the tagged telomere sequence from the streptavidin-functionalized bead using a restriction enzyme (e.g., restriction endonuclease), wherein the restriction enzyme cleaves the tagged telomere sequence at a restriction enzyme-specific site.
  • the restriction enzyme can include EcoRl or Asci. In some embodiments, the restriction enzyme can include Clal, Pvul, AsiSI, PacI, or Pmel.
  • methods described herein can produce increased telomere enrichment over standard whole genome sequencing (FIG. 3A). In some embodiments of direct telomere profding, methods described herein can achieve improved telomere length measurement as compared to conventional assays (e.g., standard whole genome sequencing). In some embodiments of direct telomere profding, improved telomere length measurement can be achieved by the Direct Telomere Profding method described herein. In some embodiments of direct telomere profding, methods described herein can reproduce telomere length measurements from telomere length measurements by Southern Blotting (FIG. 4).
  • methods described herein can produce telomere length measurements without the length bias of nanopore sequencing (FIG. 5A). In some embodiments of direct telomere profding, methods described herein can reproduce telomere profding results with lower variability compared to other sequencing methods (FIGs. 6A and 6B).
  • methods provided herein can achieve accurate telomere length measurement using small amounts of DNA (e.g., smaller than would conventionally be required).
  • about 5 to about 10 e.g., about 5 to about 9, about 5 to about 8, about 5 to about 7, about 5 to about 6, about 6 to about 10, about 6 to about 9, about 6 to about 8, about 6 to about 7, about 7 to about 10, about 7 to about 9, about 7 to about 8, about 8 to about 10, about 8 to about 9, or about 9 to about 10.
  • micrograms of DNA can be used to achieve accurate telomere length measurement.
  • methods provided herein can be used in multiplex format. In some embodiments of direct telomere profding, methods provided herein can be used in multiplex format for screening purposes. In some embodiments of direct telomere profding, methods provided herein can be used in multiplex format for screening purposes wherein more than 10 (e.g., more than 20, more than 30, more than 40, more than 50, more than 70, or more than 100) samples are tested simultaneously.
  • more than 10 e.g., more than 20, more than 30, more than 40, more than 50, more than 70, or more than 100
  • methods provided herein can be used in multiplex format for screening purposes wherein more than 100 (e.g., more than 200, more than 300, more than 400, more than 500, more than 600, more than 700, more than 800, more than 900, or more than 1000) samples are tested simultaneously.
  • methods provided herein can be used to assign chromosome status to the telomere reads.
  • a “probe” and “tagging probe” can refer to any molecule capable of capturing (directly or indirectly) and/or labelling an analyte (e.g., DNA molecule) in a biological sample.
  • the tagging probe is a nucleic acid or a polypeptide.
  • the tagging probe includes a barcode (e.g., a unique molecular identifier (UMI)).
  • UMI unique molecular identifier
  • a tagging probe can be coupled to a nucleic acid sequence using any one of many different techniques including, but not limited to, ligation, hybridization, and tagmentation.
  • a tagging probe can include nucleic acid sequences that add a function, e g., spacer sequences, primer sequences/sites, and/or sample barcode sequences.
  • a telomere tagging probe can include a plurality of nucleic acid sequences, wherein the plurality of nucleic acid sequences can be attached to each other via ligation of the nucleic acids.
  • methods described herein include attaching a telomere tagging probe to a 3’ end of the telomere to generate a tagged telomere sequence.
  • the attaching step comprises ligating the telomere tagging probe to the 3’ end of the telomere.
  • the ligating comprises cycling ligation.
  • the ligating comprises using a DNA ligase enzyme.
  • the DNA ligase enzyme is from a bacterium, e.g., the DNA ligase enzyme is a bacterial DNA ligase enzyme.
  • the DNA ligase enzyme is from a virus (e.g., a bacteriophage).
  • the DNA ligase can be T4 DNA ligase.
  • Other enzymes appropriate for the ligation step include, but are not limited to, Tth DNA ligase, Taq DNA ligase, Thermococcus sp. (strain 9oN) DNA ligase (9oNTM DNA ligase), and Ampligase®. Derivatives, e.g., sequence-modified derivatives, and/or mutants thereof, can also be used.
  • the ligating comprises a Taq DNA ligase.
  • the splint oligonucleotide is between 10 and 50 oligonucleotides in length (e.g., between 10 and 45, between 10 and 40, between 10 and 35, between 10 and 30, between 10 and 25, between 10 and 20, or between 10 and 15, between 15 and 50, between 15 and 45, between 15 and 40, between 15 and 35, between 15 and 30, between 15 and 25, between 15 and 20, between 20 and 50, between 20 and 45, between 20 and 40, between 20 and 35, between 20 and 30, between 20 and 25, between 25 and 50, between 25 and 45, between 25 and 40, between 25 and 35, between 25 and 30, between 30 and 50, between 30 and 45, between 30 and 40, between 30 and 35, between 35 and 50, between 35 and 45, between 35 and 40, between 40 and 50, between 40 and 45, or between 45 and 50 oligonucleotides in length).
  • a method for determining a telomere length of a DNA molecule from a biological sample can include (a) providing a telomere tagging probe, wherein the telomere tagging probe comprises a unique molecular identifier (UMI) sequence; (b) providing a splint oligonucleotide, wherein the splint oligonucleotide specifically binds to at least a portion of a telomere on the DNA molecule and to at least a portion of the telomere tagging probe; (c) attaching the telomere tagging probe to the telomere to generate a tagged telomere sequence; (d) contacting the tagged telomere sequence with a forward primer and a reverse primer, wherein the forward primer binds to a subtelomere of the telomere, and the reverse primer binds to at least a portion of the telomere
  • amplifying step (e) further comprises generating a forward amplified tagged telomere sequence and a reverse amplified tagged telomere sequence, wherein the forward amplified tagged telomere sequence is generated by an amplification reaction using the forward primer, and wherein the reverse amplified tagged telomere sequence is generated by an amplification reaction using the reverse primer.
  • the splint oligonucleotide is blocked, such that the blocked splint oligonucleotide is a splint oligonucleotide that is blocked at the 3’ end such that it cannot be extended by a nucleic acid polymerase.
  • a blocked splint oligonucleotide can include a 3’ end modification (e.g., a 3’ dideoxy C (3’ddC), 3’ddG, 3’ddA, 3’ddT, 3’ inverted dT, 3’ C3 spacer, 3’ amino, 3’ biotinylation, or 3’ phosphorylation).
  • PacBio TeloTag Mulitplex Adapters with UMI - one of these multiplexing primers can be used with the splints above.
  • a 1:100 dilution for the annealed adapters mix was made in lx hifi TAQ buffer.
  • the taq buffer was heated and then diluted lOpl of buffer with 90 pL of water.
  • the solution was cooled down by placing on ice.
  • 99pL of this IX hifi Buffer was taken and IpL of the annealed adapter mix was added.
  • the components were then mixed in a protein lo-bind tube on ice and the tubes were placed in thermocycles and a cycling ligation program was run, wherein the program heats to 70°C and denatures the DNA just enough to make sure the 3' telomere overhang is not forming any secondary structures.
  • the thermocycle then slowly cools to 45°C where the ligation takes place. This was repeated for 15 cycles to allow for binding of the different permutations of the telomere adapter to capture all telomeres.
  • the restriction enzyme used is a sequence that should not be either in the subtelomere or the adapters that are ligated. EcoRl was used, since EcoRl is compatible with the adapters listed above however other appropriate restriction enzymes could be used instead. 3 pl of EcoRl HF (100,000 U/mL) was added to the purified tagged DNA in cutsmart and digested for 2 hours at 37°C.
  • Input DNA should be around 20 ng and never more than 100 ng as it can inhibit PCR. It was important to use Failsafe polymerase as other polymerases cannot amplify through the telomeric end.
  • telomeres are expected to have widely different lengths: equimolar amounts are needed for each DNA sample. It is needed to cleanup each sample separately, quantitate the DNA, calculate equimolar concentrations and then pool the samples. If telomeres are around the same length: the samples can just be pooled at this step all in one tube.
  • the AMPure PB beads and DNA were mixed and the tube was placed on hula at rotating at lOrpm for 7 mins. The tube was then placed on magnet for 1:30 mins, and then washed 2X with freshly made 80% ethanol. Then, the tube was taken off magnet and eluted with warmed and well mixed PacBio Elution buffer (55pL per library prep reaction). The sample was removed from magnet and mix gently with normal rainin pipette tips (not wide orifice) for 20 times to break up the bead/DNA complex. The pooled samples were incubated at 37°C for 15 mins and placed on magnet for 2 min. A P200 rainin tip was used to suck up the liquid slowly and place on protein low bind tubes, where the amount of DNA was measured using Qubit HS (lul of DNA).
  • the DNA Prep Additive was prepared.
  • the DNA Prep Additive was diluted with Enzyme Dilution Buffer to a total volume of 5 pL. 10.0 pL of the master mix was added to the tube-strips containing 45.0 pL - 53.0 pL of sheared DNA. The total volume in this step was 55.0 pL - 63.0 pL.
  • a wide bore pipette was used to mix the reaction well by pipetting up and down 20 times, and the contents of the tube strips were then spun down with a quick spin in a microfuge.
  • DNA Damage Repair Mix was added to repair DNA damage, then the samples were treated with End Prep Mix for DNA end repair and A tailing.
  • Adapter ligation was performed by adding Overhang Adapter to each sample, followed by size select of SMRTbell library using 1.0X AMPure® PB beads, wherein AMPure PB beads were added to the ligation reaction and the DNA was then eluted with elution buffer.
  • the libraries were then sequenced with PacBio’ s HiFi read technology. PacBio HiFi reads rely on extended run times (30 hours +) to allow ample time for the sequencing polymerase to pass through circularized DNA multiple times (Median of 23 passes in a representative run).
  • the adapters include a set of 6 splints, each with one of the 6 permutations for the telomere register 3x(CCCTAA).
  • the 3x(CCCTAA) complementary region binds to the 3' overhang found in all telomeres, while the unique adapter region is a perfect complement (splint) to the biotin adapter that is then ligated to the G strand telomere.
  • Adapters for chromosome specific pull downs (Cutting with an 8-base cutter (Asci) to release from beads leaving ⁇ 64kb of subtelomere) are also shown below.
  • NB 16 set
  • the oligos were annealed, then first denatured at 95°C and cooled by decreasing the temperature by 1 Degree°C/min until the temperature reached predicted Tm (85°C). The adapter mix was then held at the Tm for 10 mins. After this, the adapter mix was cooled to 4°C by cooling 1°C/ min. The annealed adapters were then held at 4 degrees.
  • the streptavidin beads cannot bind to HMW DNA, it was needed to partially digest the DNA.
  • the type of restriction enzyme used depends on how much of the subtelomere that would need to be captured.
  • BamHI was used that leaves ⁇ 4kb of subtelomere sequence.
  • EcoRI was used to release the adapters from streptavidin beads at a later step.
  • an 8 base cutter can be used (e.g., Notl or Asci) with the biotin adapters that have a Notl or Asci cut site instead.
  • the tradeoff here is that fewer molecules are pulled down compared to the 6bp cutter due to the properties of the streptavidin beads.
  • the taq buffer was heated and then diluted lOpl of buffer with 90 pL of water.
  • the solution was cooled down by placing on ice.
  • 99pL of this IX hifi Buffer was taken and I pL of the annealed adapter mix was added.
  • the components were then mixed in a protein lo-bind tube on ice and the tubes were placed in thermocycler and a cycling ligation program was run, wherein the program heats to 70°C and denatures the DNA just enough to make sure the 3' telomere overhang is not forming any secondary structures.
  • the thermocycler then slowly cools to 45°C where the ligation takes place. This was repeated for 15 cycles to allow for binding of the different permutations of the telomere adapter to capture all telomeres.
  • the tube was then placed on magnet for 1 hr and 30 min. The supernatant very gently pipetted off and washed 2X with freshly made 85% ethanol. The Ethanol was taken off, spun down using a microcentrifuge, and placed back on magnet and any trace amounts of ethanol was removed using a P20 pipette. 200 pl (per 40 pg of input DNA) of the warmed IX cutsmart was added, the tube was then removed from magnet and mix gently with rainin pipette tip (not wide orifice) for 20 times to break up the bead/DNA complex. The pooled samples were incubated at 37°C for 15 mins and placed on magnet for 2 min. The amount of DNA was then measured using Qubit BR (Ipl of DNA).
  • the eppendorf tube was placed on a hula (taped or rubber banded horizontally with a 15-30 degree slight slant) and incubated at room temp for 20mins at Irpm. The samples were placed on the magnet for 1 min or until the supernatant seems clear, then removed from magnet and added on top of beads 1ml of DynaBeads high salt Washing Solution. The samples were placed on the hula or roller to continue resuspension and washing for 5 mins.
  • a digest solution was prepared to release the telomeres from the streptavidin beads, where the beads were placed back on magnet and 75pl of digest solution was added to the beads. If multiple samples are being multiplexed for sequencing this is the step to pool them and do a pooled digestion: resuspend 1 sample and then move the bead/digest solution from tube 1 to the next tube to pool and digest the samples together. The samples were incubated for 2h taped horizontally with a slight angle on hula inside the 37°C incubator rotating at 2 rpm. The beads were then placed on magnet and the supernatant contains released telomeres.
  • the DNA from the digest step was measured using Qubit HSyour DNA, where a total DNA range of about 6-20ng of DNA was measured in the lo-bind tube.
  • telomere length is maintained by telomerase around an equilibrium length distribution. To accurately measure telomere length, sequence analysis requires many telomere reads to capture the full distribution of lengths. For yeast, whole genome sequencing using nanopore profding generated hundreds of reads per telomere. For human DNA, however, whole genome sequencing generated relatively few telomere reads.
  • telomere enrichment method was developed that generates thousands of telomere reads per sample (FIG. 3A).
  • High molecular weight DNA was prepared from between 5-40mg of DNA and ligated a splinted biotinylated oligonucleotide (TeloTag) to all telomeres. Cutting the DNA with restriction enzyme allowed efficient enrichment of telomere fragments using streptavidin beads. The optimal ratio of streptavidin beads to TeloTagged input genomic DNA was determined, which can capture -15-20% of all input telomeres as determined by comparison to Southern blot (FIG. 5B).
  • the TeloTag includes a barcode for multiplexing samples and a restriction site to release the bound fragments from the streptavidin beads.
  • telomere length was calculated as the number of base pairs between the TeloTag and the subtelomere - telomere boundary (FIG. 3B).
  • the human telomere subtelomere contains many “variant” telomere repeats such as TGAGGG, TCAGGG that differ from the canonical TTAGGG sequence due to mutation accumulation over time.
  • the subtelomere boundary was set as the position where there is a significant deviation from the TTAGGG repeat base composition using a rolling window moving from the telomere into the subtelomere. This method differs from previous studies using 2X TTAGGG as the boundary as it incorporates the variant repeats into the telomere since they may contribute to the length regulation.
  • telomere length may affect the calculated telomere length, especially due to errors on the strand that contains CCCTAA repeats (C strand).
  • C strand CCCTAA repeats
  • the median difference in length was 344 bp (FIG. 5A). (The length differences were greater on longer telomere suggesting a systematic error in base calling).
  • the electrical signal was examined in the Fast5 fdes. An algorithm was established to count the repeated TTAGGG peaks in the signal data and found a good correlation of the number of repeats with telomere length determined by Guppy base-calling.

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Abstract

L'invention concerne des compositions et des procédés permettant de déterminer une longueur de télomère. Dans certains modes de réalisation, les procédés comprennent la fixation d'une sonde de marquage de télomère à une extrémité 3' du télomère pour générer une séquence de télomère marquée et l'utilisation d'un oligonucléotide d'attelle comprenant une séquence d'acides nucléiques qui se lie spécifiquement à au moins une partie de la sonde de marquage de télomère et à au moins une partie du télomère.
EP23861627.0A 2022-09-01 2023-09-01 Procédés de mesure de longueur de télomère Pending EP4581367A1 (fr)

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