Increased yields of duplex sequencing data by a series of quality control tools

Povysil, Gundula; Salazar, Renato; Stoler, Nicholas; Nekrutenko, Anton; Tiemann‐Boege, Irene

doi:10.1093/nargab/lqab014

Cited by 5 publications

(11 citation statements)

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“…Consensus sequences were first formed using Du Novo (Stoler et al 2016;Stoler et al 2020) and variants were called using the Naive Variant Caller (Blankenberg et al 2014), followed by a more thorough screening with our Variant Analyzer software (Povysil et al 2021). The Variant Analyzer reexamines raw reads from Du Novo variant calls and provides different summary data that categorizes the confidence level of a variant by a tier-based system.…”

Section: Substitutions Detected In the Coding Region Of Fgfr3 In Sperm Dnamentioning

confidence: 99%

“…Analyzer supports the analysis of reads without a family as demonstrated in (Povysil et al 2021), which allowed maximizing the data output.…”

Section: Substitutions Detected In the Coding Region Of Fgfr3 In Sperm Dnamentioning

confidence: 99%

“…according to a DS specific pipeline that includes an error correction tool (Stoler et al 2020). Variants (substitutions only) were further inspected and assigned to tiers using the Variant Analyzer (Povysil et al 2021). Every variant was categorized as a SNP if detected in all libraries that shared the same donor pool/target region combination with a frequency ~10%.…”

Section: Data Processing and Variant Filteringmentioning

confidence: 99%

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Discovery of an unusual high number ofde novomutations in sperm of older men using duplex sequencing

Salazar

Arbeithuber

Ivankovic

et al. 2021

Preprint

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De novo mutations (DNMs) are an important player in heritable diseases and evolution, yet little is known about the different mutagenic processes in our germline given the difficulty to reliably identify ultra-low frequency variants. Of particular interest are highly recurrent DNMs associated with congenital disorders that have been described as selfish mutations expanding in the male germline, thus becoming more frequent with age. Here, we have adapted duplex sequencing (DS), an ultra-deep sequencing method that renders sequence information on both DNA strands; thus, one mutation can be reliably called in millions of sequenced bases. With DS, we examined ~4.5 kb of the FGFR3 coding region in sperm DNA from older and younger donors. We identified highly mutable sites with mutation frequencies 4-5 orders of magnitude higher than the genome average. Multiple mutations were found at a higher frequency, or exclusively, in older donors, suggesting that these mutations are testis exclusive mosaics expanding in the male germline with age. Also, older donors harbored more mutations associated with congenital disorders. Some mutations were found in both age groups with no significant difference, suggesting that these might result from a different mechanism (e.g., post-zygotic mosaicism). We also observed that independently of age, the frequency and deleteriousness of the mutations in sperm were elevated compared to reports in the population. Our approach is an important strategy to identify mutations that could be associated with aberrant receptor tyrosine kinase activity, with unexplored consequences in a society with delayed fatherhood.

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Section: Substitutions Detected In the Coding Region Of Fgfr3 In Sperm Dnamentioning

confidence: 99%

“…Analyzer supports the analysis of reads without a family as demonstrated in (Povysil et al 2021), which allowed maximizing the data output.…”

Section: Substitutions Detected In the Coding Region Of Fgfr3 In Sperm Dnamentioning

confidence: 99%

Section: Data Processing and Variant Filteringmentioning

confidence: 99%

See 1 more Smart Citation

Discovery of an unusual high number ofde novomutations in sperm of older men using duplex sequencing

Salazar

Arbeithuber

Ivankovic

et al. 2021

Preprint

Self Cite

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show abstract

“…Variant calling was then performed by the variant caller LoFreq. Finally, the variants (substitutions only) were further inspected and assigned to tiers using the Variant Analyzer 60 . Variants with DCS coverage below 500 and variants outside the probe regions were discarded from our analysis and only Tier 1 variants were kept, together with Tier 2 that were detected more than once.…”

Section: Duplex Sequencing Data Analysismentioning

confidence: 99%

“…Variants with DCS coverage below 500 and variants outside the probe regions were discarded from our analysis and only Tier 1 variants were kept, together with Tier 2 that were detected more than once. For more details on this analysis see Povysil et al 60 . The full Galaxy workflow is publicly available: https://usegalaxy.org/u/jku-itb-lab/w/gdansk-paper---galaxy-workflow.…”

Section: Duplex Sequencing Data Analysismentioning

confidence: 99%

High prevalence of somatic PIK3CA and TP53 pathogenic variants in the normal mammary gland tissue of sporadic breast cancer patients revealed by duplex sequencing

Kostecka

Nowikiewicz

Olszewski

et al. 2021

Preprint

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The mammary gland undergoes hormonally stimulated cycles of proliferation, lactation and involution. We hypothesized that these factors increase the mutational burden in glandular tissue and may explain high cancer incidence rate in the general population and recurrent disease. Hence, we investigated the DNA sequence variants in the normal mammary gland, tumor and peripheral blood from 52 reportedly sporadic breast cancer patients, including breast-conserving surgery cases. Targeted resequencing of 542 cancer associated genes revealed mosaic somatic pathogenic variants of: PIK3CA, TP53, AKT1, MAP3K1, CDH1, RB1, NCOR1, MED12, CBFB, TBX3 and TSHR in the normal mammary gland, at considerable allelic frequencies (9x10-2 to 5.2x10-1) indicating clonal expansion. Further evaluation of the frequently damaged PIK3CA and TP53 genes by ultra-sensitive duplex sequencing demonstrated a diversified picture of multiple low level-mosaic (in 10-2 to 10-4 alleles) hotspot pathogenic variants. Our results raise a question about the oncogenic potential in non-tumor mammary gland tissue of breast-conserving surgery patients.

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High prevalence of somatic PIK3CA and TP53 pathogenic variants in the normal mammary gland tissue of sporadic breast cancer patients revealed by duplex sequencing

et al. 2022

Self Cite

View full text Add to dashboard Cite

The mammary gland undergoes hormonally stimulated cycles of proliferation, lactation, and involution. We hypothesized that these factors increase the mutational burden in glandular tissue and may explain high cancer incidence rate in the general population, and recurrent disease. Hence, we investigated the DNA sequence variants in the normal mammary gland, tumor, and peripheral blood from 52 reportedly sporadic breast cancer patients. Targeted resequencing of 542 cancer-associated genes revealed subclonal somatic pathogenic variants of: PIK3CA, TP53, AKT1, MAP3K1, CDH1, RB1, NCOR1, MED12, CBFB, TBX3, and TSHR in the normal mammary gland at considerable allelic frequencies (9 × 10−2– 5.2 × 10−1), indicating clonal expansion. Further evaluation of the frequently damaged PIK3CA and TP53 genes by ultra-sensitive duplex sequencing demonstrated a diversified picture of multiple low-level subclonal (in 10−2–10−4 alleles) hotspot pathogenic variants. Our results raise a question about the oncogenic potential in non-tumorous mammary gland tissue of breast-conserving surgery patients.

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Increased yields of duplex sequencing data by a series of quality control tools

Cited by 5 publications

References 0 publications

Discovery of an unusual high number ofde novomutations in sperm of older men using duplex sequencing

Discovery of an unusual high number ofde novomutations in sperm of older men using duplex sequencing

High prevalence of somatic PIK3CA and TP53 pathogenic variants in the normal mammary gland tissue of sporadic breast cancer patients revealed by duplex sequencing

High prevalence of somatic PIK3CA and TP53 pathogenic variants in the normal mammary gland tissue of sporadic breast cancer patients revealed by duplex sequencing

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