Failure to detect mutations in<i>U2AF1</i>due to changes in the GRCh38 reference sequence

Miller, Christopher A.; Walker, Trent; Tl, Jensen; Wf, Hooper; Rs, Fulton; Js, Painter; Ma, Sekeres; Tj, Ley; Dh, Spencer; Jb, Goll; Mj, Walter

doi:10.1101/2021.05.07.442430

Cited by 4 publications

(4 citation statements)

References 16 publications

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“…While previous studies concluded that variant calling performance is generally better on GRCh38 34 , 35 , our benchmark demonstrates that variant calls in some genes are less accurate on GRCh38 than GRCh37. Another group recently independently identified the importance of masking the extra copy of one gene ( U2AF1/U2AF1L5 ) for cancer research 36 . Our results identify that false duplications cause many of the discrepancies found recently between exome variant calls on GRCh37 and GRCh38, 37 .We produced similar benchmarks for both versions of the reference, so that scientists can better understand strengths and weaknesses of each reference, and test modifications to the reference such as the hs37d5 decoy for GRCh37 or the masked GRCh38 we propose here.…”

Section: Discussionmentioning

confidence: 99%

Curated variation benchmarks for challenging medically relevant autosomal genes

et al. 2022

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The repetitive nature and complexity of some medically relevant genes poses a challenge for their accurate analysis in a clinical setting. The Genome in a Bottle Consortium has provided variant benchmark sets, but these exclude nearly four hundred medically relevant genes due to their repetitiveness or polymorphic complexity. Here we characterize 273 of these 395 challenging autosomal genes using a haplotype-resolved whole-genome assembly. This curated benchmark reports over 17,000 single nucleotide variations, 3,600 INDELs, and 200 structural variations each for human genome reference GRCh37 and GRCh38 across HG002. We show that false duplications in either GRCh37 or GRCh38 result in reference-specific, missed variants for short- and long-read technologies in medically relevant genes including CBS , CRYAA , and KCNE1 . When masking these false duplications, variant recall can improve from 8% to 100%. Forming benchmarks from a haplotype-resolved whole-genome assembly may become a prototype for future benchmarks covering the whole genome.

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Section: Discussionmentioning

confidence: 99%

Curated variation benchmarks for challenging medically relevant autosomal genes

et al. 2022

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“…However, we demonstrate that masking false duplications on GRCh38 greatly improves performance in these genes. Interestingly, another group recently independently identified the importance of masking the extra copy of one gene (U2AF1/U2AF1L5) for cancer research 36 . Our results identify that false duplications cause many of the discrepancies found recently between exome variant calls on GRCh37 and GRCh38, 37 and highlight the importance of our proposed masked GRCh38 genome.…”

Section: Discussionmentioning

confidence: 99%

Towards a Comprehensive Variation Benchmark for Challenging Medically-Relevant Autosomal Genes

Wagner

Olson

Harris

et al. 2021

Preprint

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The repetitive nature and complexity of multiple medically important genes make them intractable to accurate analysis, despite the maturity of short-read sequencing, resulting in a gap in clinical applications of genome sequencing. The Genome in a Bottle Consortium has provided benchmark variant sets, but these excluded some medically relevant genes due to their repetitiveness or polymorphic complexity. In this study, we characterize 273 of these 395 challenging autosomal genes that have multiple implications for medical sequencing. This extended, curated benchmark reports over 17,000 SNVs, 3,600 INDELs, and 200 SVs each for GRCh37 and GRCh38. We show that false duplications in either GRCh37 or GRCh38 result in reference-specific, missed variants for short- and long-read technologies in medically important genes including CBS, CRYAA, and KCNE1. Our proposed solution improves variant recall in these genes from 8% to 100%. This benchmark will significantly improve the comprehensive characterization of these medically relevant genes and guide new method development.

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“…A special approach was required to identify somatic variants in U2AF1 since an erroneous segmental duplication in the region of the gene in the hg38 reference genome resulted in a mapping score of zero during alignment of the FASTQ file 50 . We developed a Rust-HTSLIB binary (https://github.com/weinstockj/pileup_region) to specifically identify reads associated with the U2AF1 variants S34F, S34Y, R156H, Q157P, and Q157R.…”

Section: Methodsmentioning

confidence: 99%

Clonal hematopoiesis is driven by aberrant activation of TCL1A

Weinstock

Gopakumar

Burugula

et al. 2021

Preprint

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A diverse set of driver genes, such as regulators of DNA methylation, RNA splicing, and chromatin remodeling, have been associated with pre-malignant clonal expansion of hematopoietic stem cells (HSCs). The factors mediating expansion of these mutant clones remain largely unknown, partially due to a paucity of large cohorts with longitudinal blood sampling. To circumvent this limitation, we developed and validated a method to infer clonal expansion rate from single timepoint data called PACER (passenger-approximated clonal expansion rate). Applying PACER to 5,071 persons with clonal hematopoiesis accurately recapitulated the known fitness effects due to different driver mutations. A genome-wide association study of PACER revealed that a common inherited polymorphism in the TCL1A promoter was associated with slower clonal expansion. Those carrying two copies of this protective allele had up to 80% reduced odds of having driver mutations in TET2, ASXL1, SF3B1, SRSF2, and JAK2, but not DNMT3A. TCL1A was not expressed in normal or DNMT3A-mutated HSCs, but the introduction of mutations in TET2 or ASXL1 by CRISPR editing led to aberrant expression of TCL1A and expansion of HSCs in vitro. These effects were abrogated in HSCs from donors carrying the protective TCL1A allele. Our results indicate that the fitness advantage of multiple common driver genes in clonal hematopoiesis is mediated through TCL1A activation. PACER is an approach that can be widely applied to uncover genetic and environmental determinants of pre-malignant clonal expansion in blood and other tissues.

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Failure to detect mutations inU2AF1due to changes in the GRCh38 reference sequence

Cited by 4 publications

References 16 publications

Curated variation benchmarks for challenging medically relevant autosomal genes

Curated variation benchmarks for challenging medically relevant autosomal genes

Towards a Comprehensive Variation Benchmark for Challenging Medically-Relevant Autosomal Genes

Clonal hematopoiesis is driven by aberrant activation of TCL1A

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