• evo视讯(中国)

    Tn5-Duplex-Sequencing (Tn5-Duplex-Seq) for low-input single-molecule variant detection

    Diane Shao, Nazia Hilal, Sangita Choudhury

    Published: 2024-02-02 DOI: 10.17504/protocols.io.6qpvr3nbzvmk/v1
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    Abstract

    DNA mutations are the inevitable consequences of errors that arise during replication-repair of DNA damage as well as aging and disease progression. Because of their random and infrequent occurrence, quantification, and characterization of DNA mutations in the genome of somatic cells have been difficult. These mutations in DNA drive genetic diversity, alter gene function, define evolutionary trajectories, and provide targets for precision medicine and diagnostics. It is crucial to detect mutations across a wide range of abundance, i.e., variant allele frequency (VAF). Detecting low-abundance mutations (e.g., <0.1–1% VAF or in individual cells) is important for understanding human embryonic development, somatic mosaicism, and clonal hematopoiesis and uncovering pathogenic variants. Altogether somatic mutations provide important and unique insights into the biology of complex diseases. To decipher the causal inference, we must build robust genetic maps of somatic evolution in health and disease. The recent advent of duplex consensus sequencing has heralded a new generation of accuracy. However, multiple techniques focus on targeted areas of the genome (Twin Strand Biosciences) or are limited to restriction sites (Nanoseq), limiting their application to comprehensive somatic variant characterization. Furthermore, fragmentation of the genome and standard A-tailing and ligation creates errors (BotseqS, CODEC). Ligation of duplex strands for efficient sequencing has proven promising, though in practice requires complex molecular structures (Pro-Seq, CODEC) which have been observed to frequently result in incorrectly paired duplexes (CODEC). To enable comprehensive variant detection by next-generation DNA sequencing, we propose an innovative, accessible, and highly accurate Tn5 transposase-based duplex-sequencing technology ( Tn5-duplex-seq ) where complementary strands of DNA could be labeled at the molecular level in a single-tube reaction; thus, identifying single nucleotide variants (SNVs) from single-molecules of DNA A regardless of starting from single cells or pooled cell/DNA input. . The conceptual basis of the protocol comes from META-CS (Xing et al.2021), a Tn5 based aproach optimized for single-cell whole genome amplification. We find that modifications of this approach to include flexible input and the sequencing strategy to optimize cost per variant detection enables great flexibility for all low-input applications.

    Tn5-duplex-seq approach offers several benefits over other duplex approaches including.

    (1) preservation of original template molecules by utilizing 16 unique sequences (Compared to the loss of 50% of

    molecules due to intramolecular symmetry during TN5-based Nextera library preparation)

    (2) accuracy by eliminating the requirement for A-tailing

    (3) efficiency of duplex capture through specifying input

    (4) accessibility by using standard reagents and oligonucleotide preparations

    (5) distinction between double-stranded SNVs and single-stranded lesions.

    Our method enables library preparation for short-read sequencing. Downstream analysis enables accurate and high-throughput SNV/indel and copy number analysis.

    Steps

    PROTOCOL MATERIALS

    1.

    100millimolar (mM)

    10millimolar (mM)

    20mg/mL

    RECIPE FOR MAKING IN-HOUSE REAGENTS

    2.

    2X Single Cell Lysis Buffer

    40millimolar (mM)

    40millimolar (mM)

    0.3% volume

    Note
    Make 1X Single Cell Lysis Buffer on the day of sort (30 reactions) Add TL Proteinase K + 1M DTT to of 2X cell lysis buffer. Dilute 1:1 with water to obtain 1X solution for sorting directly into the buffer.

    12X quenching solution

    600millimolar (mM)

    90millimolar (mM) 0.02% volume

    Note
    *Before use prepare 6X Stop Mix for use for 20 reactions below: Dilute 1μL of TL proteinase K + 19μL PBS Add 20μL 12X stop solution for the final 6X Stop solution.
    ADP1 and ADP2 Mix

    1. Reconstitute the 16 ADP1 and 16 ADP2 primers separately in low TE and store in aliquots at -80°C until ready for use.
    2. Make an equimolar mix of the 16 ADP1 and ADP2 primers to make the ADP1 and ADP2 mix respectively.

    Note
    each primer x 16 primers for total solution

    TRANSPOSOME LOADING

    3.

    Transposon Annealing

    • Reconstitute 16 META-CS oligos and 1 reverse oligo to 100micromolar (µM) in Annealing Buffer (40millimolar (mM)Tris-HCl (8), 50millimolar (mM) NaCl)
    • Combine 1:1 of a singular META-CS oligo with the reverse oligo (there should be 16 separate reactions to put on the thermocycler). Mix up the reaction, spin it down briefly, and run the thermocycler using the conditions below:

    Transposon Assembly.

    • Combine all 16 reactions into one tube and aliquot for storage at -80°C.
    • Take 10μLof this aliquot and combine it with 10μL of unloaded Transposome
    • Incubate at 23°C for0h 30m 0s
    • Add 10μL of 100% glycerol.
    • Aliquot and store at -80°C.
      Note
      Estimated final concentration including glycerol storage (~16.7 μM dimerized Tn5) *Prior to use, dilute Tn5 in Diagenode Tn5 dilution buffer depending on the desired concentration Optimization of Tn5 concentration: Check on 50 cells using dilutions of 1:500, 1:750, 1:1000, and 1:1500, and check the tagmentation curve.

    TN5-DUPLEX LIBRARY PROCEDURE

    4.

    Sorting and lysing cells 2μL

    • Prepare nuclei for sorting.
    • Sort cells directly into 2μL of 1X cell lysis buffer Run the thermocycler using the conditions below 65°C

    for for hold The plate can be stored after lysis.

    5.

    Tn5 tagmentation 8μL

    • Add 8μL transposition mix (total 10μL reaction). Vortex, spin down.
    • 5μLDiagenode 2X Tagmentation buffer
    • 1μL diluted Tn5 per optimized dilution instructions above
    • 2μL H2O Incubate in thermocycler using the conditions below 65°C

    for hold

    6.

    Quenching 2μL

    • Prepare 6X Stop Mix and add 2μL Mix per tube. Spin down, vortex, and spin down.
    • Incubate in thermocycler using the conditions below 65°C

    for for hold

    7.

    First Strand tagging 13μL

    Add 13μL Strand Tagging Mix 1. Vortex and spin down.

    • 5μL Q5 Reaction Buffer
    • 5μL μL Q5 High GC Enhancer
    • 0.6μL 100millimolar (mM) MgCl2
    • 0.5μL 10millimolar (mM) dNTP mix
    • 0.25μL BSA 20mg/ml
    • 0.25μL Q5 polymerase
    • 0.85μL``100micromolar (µM) ADP1 primer mix
    • 0.55μL H2O Incubate in thermocycler using the conditions below 105°C

    for
    for     for for hold

    8.

    Stop reaction 1μL

    Add 1μL Thermolabile ExoI per tube. Try to touch the minimum of the solution surface. Spin down first, then plate mix, and spin down again.

    for for hold

    9.

    Second Strand tagging 4μL

    Add 4μL Strand Tagging 2 Mix (total 30μL). Vortex and spin down.

    • 1μL Q5 Reaction Buffer
    • 1μL Q5 High GC Enhancer
    • 0.95μL 100micromolar (µM) ADP2 primer mix
    • 0.1μL 10millimolar (mM) each dNTP mix
    • 0.1μL Q5 polymerase
    • 0.85μL H2O Incubate in thermocycler using the conditions below 105°C.

    for for for hold

    10.

    Stop reaction 1μL

    Add 1μL Thermolabile ExoI per tube. Try to touch the minimum of the solution surface. Spin down first, then plate mix, and spin down again.

    for 37°C for 0h 15m 0s for 65°C for0h 5m 0s hold

    11.

    Library prep 14μL

    1. Make PCR Mix (per cell):
    • 5μL NEB Universal Primer (NEB E7335S, E7500S, E7710S, E7730S)
    • 4μL Q5 Reaction Buffer
    • 4μLQ5 High GC Enhancer
    • 0.4μL 10millimolar (mM) each dNTP mix
    • 0.4μL water (H2O)
    • 0.2μL Q5 polymerase *add last 2.Add 5μL NEB Index Primer per tube, avoiding touching the liquid.

    3.Add 14μL PCR Mix per tube, avoiding touching the liquid. Vortex and spin down.

    1. Incubate in thermocycler using the conditions below

    for 12 cycles of for , for for hold

    PURIFICATION AND ZYMO CLEAN

    12.

    Zymo clean

    1. Utilize the Zymo DNA Clean & Concentrator Kit with associated protocol (abbreviated version below).
    • For microbulk samples, use 4:1 DNA binding buffer to sample (200μL buffer to 50μLreaction). For single cell samples, pool desired samples first, then measure the total pooled volume and use 4:1 DNA binding buffer to sample volume. For single cell samples, pool 5 cells per spin column. For 50 cell samples, use 1 spin column per sample.
    • Add it to the spin column. The maximum volume that the spin column can hold is 800μL so pooled samples should have to be run through the same column sequentially until all of the liquid has been run through, discarding flowthrough each time.
    • Spin for 0h 0m 30s at maximum speed on the tabletop centrifuge >10,000xg at RT
    • Add 200μL wash buffer (with ethanol added) and centrifuge column
    • Repeat wash again
    • Add 42μL x0.1 TE to elute and wait 0h 4m 0s at room temperature
    • Spin for 0h 0m 30s at maximum speed on the tabletop centrifuge >10,000xg at RT
    1. Run 2μL on High Sensitivity D5000 TapeStation chip.
    13.

    Size Selection (AMPure) 40μL

    1. Add (0.55X) resuspended AMPure XP beads to DNA library 22μL(0.55X) resuspended AMPure XP beads to 40μL DNA library. Vortex and spin down. Label the tube as “A”. Incubate for 0h 5m 0s at RT.
    2. Place tube A on a magnetic stand for 0h 5m 0s. Carefully transfer the supernatant to a new tube. Label the new tube as “B”.
    3. Size select tube “A” (0.55x AMPure XP beads): a. Add 200μL of 80% freshly prepared ethanol to all tubes while in the magnetic stand, then carefully remove and discard the supernatant.

    b. repeat the ethanol wash step one more time.

    c. Let air dry on magnetic stand for 0h 1m 0s at RT.

    d. Remove the tubes from the magnetic stand. Elute DNA from beads with 12μL 0.1X TE (for

    single cell pools) or18μL 0.1X TE (for 50 cell pools). Vortex and gently spin down, incubate for 3 min at RT.

    e. Place the tubes on the magnetic stand for 0h 3m 0s. Transfer 18μL of supernatant to a clean tube.

    1. Size select tube "B" (0.8x AMPure XP beads): a. Add AMPure XP beads to DNA solution in tube B (0.15X) 8μL AMPure XP beads to DNA solution in tube B (0.15X). Vortex

    and gently spin down. Incubate for0h 5m 0s at RT.

    b. Place tube B on the magnetic stand for 0h 5m 0s. Remove supernatant from tube B.  

    i. IMPORTANT: save the supernatant in case AmPure did not work! (the DNA will still be in the supernatant)

    c. Add 200μL of 80% freshly prepared ethanol to all tubes while in the magnetic stand, then carefully remove and discard the supernatant.

    d. repeat the ethanol wash step one more time.

    e. Let air dry on magnetic stand for 1 min at RT.

    f. Remove the tubes from the magnetic stand. Elute DNA from beads with 12μL 0.1X TE (for

    single cell pools) or18μL 0.1X TE (for 50 cell pools). Vortex and gently spin down, incubate for 3 min at RT.

    g. Place the tubes on the magnetic stand for 0h 3m 0s. Transfer 18μL of supernatant to a clean tube.

    1. Run 2μLon High Sensitivity D5000 TapeStation chip. (Run1μL of sample and 1 of 0.1x TE instead of 2μL of sample if you need to preserve sample). (Expected concentration for 5 single cell pools is: 300-1000pg/μL. Expected concentration for 50 cell pools is: 2000-15000pg/μL .
    2. Proceed to DNA quantification with final product and dilute accordingly for sequencing. Run TapeStation, need at least 5nanomolar (nM) concentration for sequencing. *Fraction B yields best sequencing results.

    <img src="http://static.aivcloud.com/literature_test/protocol_io_true/protocols.io.6qpvr3nbzvmk/pp6dcb2up0_update" alt="-
    Tube "A" (0.55x) contains DNA fragments with an average of 1000bp. Tube "B" (0.15x) contains DNA fragments with an average of 400-500bp (fragment size may range from 300bp-600bp). " loading="lazy" title="-
    Tube "A" (0.55x) contains DNA fragments with an average of 1000bp. Tube "B" (0.15x) contains DNA fragments with an average of 400-500bp (fragment size may range from 300bp-600bp). "/>

    Appendix-1

    14.

    SI Appendix  Table S1: Oligonucleotide sequences of META-CS transposon DNA and primers.

    All oligos are HPLC purified

    ABC
    AB
    META transposon sequences:
    META-CS-1GGCACCGAAAAAGATGTGTATAAGAGACAG
    META-CS-2CTCGGCGATAAAAGATGTGTATAAGAGACAG
    META-CS-3GGTGGAGCATAAAGATGTGTATAAGAGACAG
    META-CS-4CGAGCGCATTAAAGATGTGTATAAGAGACAG
    META-CS-5AGCCCGGTTATAAGATGTGTATAAGAGACAG
    META-CS-6TCGGCACCAATAAGATGTGTATAAGAGACAG
    META-CS-7GCCTGTGGATTAAGATGTGTATAAGAGACAG
    META-CS-8GCGACCCTTTTAAGATGTGTATAAGAGACAG
    META-CS-9GCATGCGGTAATAGATGTGTATAAGAGACAG
    META-CS-10GCGTTGCCATATAGATGTGTATAAGAGACAG
    META-CS-11GGCCGCATTTATAGATGTGTATAAGAGACAG
    META-CS-12ACCGCCTCTATTAGATGTGTATAAGAGACAG
    META-CS-13CCGTGCCAAAATAGATGTGTATAAGAGACAG
    META-CS-14TCTCCGGGAATTAGATGTGTATAAGAGACAG
    META-CS-15CCGCGCTTATTTAGATGTGTATAAGAGACAG
    META-CS-16CTGAGCTCGTTTTAGATGTGTATAAGAGACAG
    META-CS-rev/5Phos/CTGTCTCTTATACACATC/3InvdT/
    Adp1 primer mix:
    META-CS-1-adp1ACACTCTTTCCCTACACGACGCTCTTCCGATCTGGCACCGAAAAAGATGTGTATAAG
    META-CS-2-adp1ACACTCTTTCCCTACACGACGCTCTTCCGATCTCTCGGCGATAAAAGATGTGTATAAG
    META-CS-3-adp1ACACTCTTTCCCTACACGACGCTCTTCCGATCTGGTGGAGCATAAAGATGTGTATAAG
    META-CS-4-adp1ACACTCTTTCCCTACACGACGCTCTTCCGATCTCGAGCGCATTAAAGATGTGTATAAG
    META-CS-5-adp1ACACTCTTTCCCTACACGACGCTCTTCCGATCTAGCCCGGTTATAAGATGTGTATAAG
    META-CS-6-adp1ACACTCTTTCCCTACACGACGCTCTTCCGATCTTCGGCACCAATAAGATGTGTATAAG
    META-CS-7-adp1ACACTCTTTCCCTACACGACGCTCTTCCGATCTGCCTGTGGATTAAGATGTGTATAAG
    META-CS-8-adp1ACACTCTTTCCCTACACGACGCTCTTCCGATCTGCGACCCTTTTAAGATGTGTATAAG
    META-CS-9-adp1ACACTCTTTCCCTACACGACGCTCTTCCGATCTGCATGCGGTAATAGATGTGTATAAG
    META-CS-10-adp1ACACTCTTTCCCTACACGACGCTCTTCCGATCTGCGTTGCCATATAGATGTGTATAAG
    META-CS-11-adp1ACACTCTTTCCCTACACGACGCTCTTCCGATCTGGCCGCATTTATAGATGTGTATAAG
    META-CS-12-adp1ACACTCTTTCCCTACACGACGCTCTTCCGATCTACCGCCTCTATTAGATGTGTATAAG
    META-CS-13-adp1ACACTCTTTCCCTACACGACGCTCTTCCGATCTCCGTGCCAAAATAGATGTGTATAAG
    META-CS-14-adp1ACACTCTTTCCCTACACGACGCTCTTCCGATCTTCTCCGGGAATTAGATGTGTATAAG
    META-CS-15-adp1ACACTCTTTCCCTACACGACGCTCTTCCGATCTCCGCGCTTATTTAGATGTGTATAAG
    META-CS-16-adp1ACACTCTTTCCCTACACGACGCTCTTCCGATCTCTGAGCTCGTTTTAGATGTGTATAAG
    Adp2 primer mix:
    META-CS-1-adp2GACTGGAGTTCAGACGTGTGCTCTTCCGATCTGGCACCGAAAAAGATGTGTATAAG
    META-CS-2-adp2GACTGGAGTTCAGACGTGTGCTCTTCCGATCTCTCGGCGATAAAAGATGTGTATAAG
    META-CS-3-adp2GACTGGAGTTCAGACGTGTGCTCTTCCGATCTGGTGGAGCATAAAGATGTGTATAAG
    META-CS-4-adp2GACTGGAGTTCAGACGTGTGCTCTTCCGATCTCGAGCGCATTAAAGATGTGTATAAG
    META-CS-5-adp2GACTGGAGTTCAGACGTGTGCTCTTCCGATCTAGCCCGGTTATAAGATGTGTATAAG
    META-CS-6-adp2GACTGGAGTTCAGACGTGTGCTCTTCCGATCTTCGGCACCAATAAGATGTGTATAAG
    META-CS-7-adp2GACTGGAGTTCAGACGTGTGCTCTTCCGATCTGCCTGTGGATTAAGATGTGTATAAG
    META-CS-8-adp2GACTGGAGTTCAGACGTGTGCTCTTCCGATCTGCGACCCTTTTAAGATGTGTATAAG
    META-CS-9-adp2GACTGGAGTTCAGACGTGTGCTCTTCCGATCTGCATGCGGTAATAGATGTGTATAAG
    META-CS-10-adp2GACTGGAGTTCAGACGTGTGCTCTTCCGATCTGCGTTGCCATATAGATGTGTATAAG
    META-CS-11-adp2GACTGGAGTTCAGACGTGTGCTCTTCCGATCTGGCCGCATTTATAGATGTGTATAAG
    META-CS-12-adp2GACTGGAGTTCAGACGTGTGCTCTTCCGATCTACCGCCTCTATTAGATGTGTATAAG
    META-CS-13-adp2GACTGGAGTTCAGACGTGTGCTCTTCCGATCTCCGTGCCAAAATAGATGTGTATAAG
    META-CS-14-adp2GACTGGAGTTCAGACGTGTGCTCTTCCGATCTTCTCCGGGAATTAGATGTGTATAAG
    META-CS-15-adp2GACTGGAGTTCAGACGTGTGCTCTTCCGATCTCCGCGCTTATTTAGATGTGTATAAG
    META-CS-16-adp2GACTGGAGTTCAGACGTGTGCTCTTCCGATCTCTGAGCTCGTTTTAGATGTGTATAAG

    Indexes

    AB
    AB
    I7_Index_IDNEB index primer
    1ATCACG
    2CGATGT
    3TTAGGC
    4TGACCA
    5ACAGTG
    6GCCAAT
    7CAGATC
    8ACTTGA
    9GATCAG
    10TAGCTT
    11GGCTAC
    12CTTGTA
    13AGTCAA
    14AGTTCC
    15ATGTCA
    16CCGTCC
    17GTAGAG
    18GTCCGC
    19GTGAAA
    20GTGGCC
    21GTTTCG
    22CGTACG
    23GAGTGG
    24GGTAGC
    25ACTGAT
    26ATGAGC
    27ATTCCT
    28CAAAAG
    29CAACTA
    30CACCGG
    31CACGAT
    32CACTCA
    33CAGGCG
    34CATGGC
    35CATTTT
    36CCAACA
    37CGGAAT
    38CTAGCT
    39CTATAC
    40GTGATC
    41GACGAC
    42TAATCG
    43TACAGC
    44TATAAT
    45TCATTC
    46TCCCGA
    47TCGAAG
    48TCGGCA

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