MKT0004-2020-02 Super CHIP Duplex Sequencing ... MKT0004-2020-02 Super CHIP Duplex Sequencing...

MKT0004-2020-02 Super CHIP Duplex Sequencing ... MKT0004-2020-02 Super CHIP Duplex Sequencing Reveals
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Transcript of MKT0004-2020-02 Super CHIP Duplex Sequencing ... MKT0004-2020-02 Super CHIP Duplex Sequencing...

  • DuplexSeq™ Tag

    N ́N ́N ́N ́N ́N ́N ́N´ N N N N N N N N

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    Source DNA molecule (Truth)

    Top and bottom strands are amplified and sequenced. PCR copies are grouped

    by unique tag and strand.

    Compare top and bottom strands

    Duplex consensus eliminates errors

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    TwinStrand Duplex Sequencing™ Technology

    Sequencing Errors Obscure Truth

    Next-Generation Sequencing (NGS)

    Single Strand Error-Corrected NGS

    Duplex Sequencing

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    Abstract

    Super CHIP: Duplex Sequencing™ Reveals Clonal Dynamics in Blood that are Magnitudes more Complex than Previously Recognized Jacob Higgins, PhD1 – Gabriel Pratt, PhD1 – Charles C. Valentine, MS1 – Lindsey N Williams, PhD1 – Masumi Ueda, MD2 – Rainer Storb, MD2 Jerald P. Radich, MD2,3 – and Jesse J. Salk, MD, PhD1,3

    TwinStrand Biosciences Inc., Seattle, WA1 – Clinical Research Division, Fred Hutchinson, Seattle, WA2 – Division of Medical Oncology, University of Washington, Seattle, WA3

    • Duplex Sequencing detects multiple ultra-low frequency clonal CHIP mutations in all individuals, including the healthy 18-year old control.

    • Duplex Sequencing detects drastically more CHIP mutations per individual than in previous studies, and at much lower frequencies.

    • Background mutation frequency in the control is 4.7x10-7. • Tracking of ultra-low frequency clonal dynamics between donor and

    recipient samples reveals that there is no strong bias for clonal expansion of CHIP variants after HCT.

    • Duplex Sequencing identifies enough rare frequency variants to calculate base substitution spectra for all samples. However, there is little difference between donor and recipient spectra. The predominant base substitution signature in all samples is driven by aging.

    • The sensitivity of Duplex Sequencing may help determine whether specific CHIP mutations are associated with stronger risk of HM or cardiovascular disease.

    Conclusions

    Hematopoietic cell transplantation (HCT) is used in the treatment of hematologic malignancies (HM). Clonal hematopoiesis of indeterminate potential (CHIP) is the age-associated process whereby healthy individuals accumulate low-frequency clones driven by mutations in genes recurrently mutated in HM. CHIP is associated with increased risk of cardiovascular disease and transformation into HM.

    It has been hypothesized that CHIP clones present in HCT donors might disproportionately proliferate in the recipient due to bottlenecking and a relative growth advantage during immune reconstitution. Here we use ultra-sensitive Duplex Sequencing (DS) of genes associated with HM to investigate whether the clonal makeup of CHIP in prior donors themselves, differs from that of the engrafted donor-derived hematopoietic system multiple years after allogeneic transplant into a recipient.

    We identify dozens of mutation-bearing clones in all donor samples sequenced, which are enriched for genes previously associated with CHIP. The extremely high sensitivity and specificity of DS reveals a far more complex landscape of competing ultra-low frequency driver mutations within individuals than observed in any prior studies.

    When comparing clonal dynamics between donors and recipients we observe no significant change in clonal makeup, frequency or mutation spectrum, regardless of age. This suggests that HCT does not accelerate CHIP evolution in transplant recipients, which is reassuring from the perspective of stem cell health, secondary cancer risk, and safety of using older donors.

    Contact: https://twinstrandbio.com/contact/MKT0004 v1.0

    A DuplexSeq™ Adapter has: 1.

    2.

    Identical (or relatable) degenerate tags in each strand.

    An asymmetry allowing independent strand identification.

    For Research Use Only. Not for use in diagnostic procedures. ©2020 TwinStrand Biosciences, Inc. All rights reserved. All trademarks are the property of TwinStrand Biosciences, Inc. or their respective owners.

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    Unsupervised heirarchical clustering of the DNA base substitution spectrum in all donors, recipients, and the control. There was no strong base substitution signature unique to HCT recipients, but all donor and recipient samples show an aging signature relative to the 18-year old control. Donors and recipients ranged in age from 49-91 years old at the time of biopsy.

    No Unique Base Substitution Signature in HCT Recipients

    Total number of multi-count (clonal) variants from donors in known CHIP genes. Variants are colored by predicted effect on the canonical transcript. There was little correlation between gene size and the number of variants detected, arguing against sise-mediated bias. Compared to other approaches used in previous studies, Duplex Sequencing observed many more clonal variants in all patients and genes.

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    Dozens of multi-count (clonal) variants from known CHIP genes were identified in each donor, and only 6 in the control. Variants were colored by predicted effect on the canonical transcript. The majority were missense or nonsense, indicating in vivo selection for those that alter function of the resulting protein.

    Donor-Recipient Characteristics

    MDS

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    37.7

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    Measure of change in allele frequency in variants shared between donors and recipients. As in previous figures, variants are colored by predicted effect on the canonical transcript. Many variants increased or decreased in frequency, with the majority present at VAF < 1x10-3. Some are unique to donor or recipient (along axes), but there was not a consistent pattern of increased frequency in the recipient. This suggests that HTC does not bias age-associated expansion of clonal variants.

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    Duplex Sequencing: High Depth, Low Background

    Indel - inframe Indel - frameshift Stop Lost Start Lost Start Gained 5'-UTR

    3'-UTR Splicing Synonymous Nonsense Missense

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