Bias from removing read duplication in ultra-deep sequencing experiments

Zhou, Wanding; Chen, Tenghui; Zhao, Hao; Eterovic, Agda Karina; Meric‐Bernstam, Funda; Mills, Gordon B.; Chen, Ken

doi:10.1093/bioinformatics/btt771

Cited by 39 publications

(31 citation statements)

References 16 publications

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“…It is desirable to informatically remove artificial duplicates prior to any count or mapping-based analysis to obtain more accurate estimates of true population sizes or allele frequencies. Artificial and samplinginduced duplicates cannot be informatically separated, although the use of both mate-paired reads in the dereplication process (as performed here) can significantly improve the likelihood of removing artificial rather than sampling-induced duplicates (Zhou et al, 2014). Moreover, due to the high genomic complexity of metagenomic samples, the frequency of sampling-induced duplicates is likely to be quite low (Gomez-Alvarez et al, 2009).…”

Section: Effects Of Library Preparation On Metagenomic Library Qualitymentioning

confidence: 99%

“…2). Artificial and samplinginduced duplicates cannot be informatically separated, although the use of both mate-paired reads in the dereplication process (as performed here) can significantly improve the likelihood of removing artificial rather than sampling-induced duplicates (Zhou et al, 2014). Duplicated reads can arise from two sources: overamplification of a limited pool of input molecules ('artificial') or fragmentation of two identical genomic molecules in exactly the same place during library preparation ('sampling induced').…”

Section: Effects Of Library Preparation On Metagenomic Library Qualitymentioning

confidence: 99%

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The effects of variable sample biomass on comparative metagenomics

Chafee

Maignien

Simmons

2015

Environmental Microbiology

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Longitudinal studies that integrate samples with variable biomass are essential to understand microbial community dynamics across space or time. Shotgun metagenomics is widely used to investigate these communities at the functional level, but little is known about the effects of combining low and high biomass samples on downstream analysis. We investigated the interacting effects of DNA input and library amplification by polymerase chain reaction on comparative metagenomic analysis using dilutions of a single complex template from an Arabidopsis thaliana-associated microbial community. We modified the Illumina Nextera kit to generate high-quality large-insert (680 bp) paired-end libraries using a range of 50 pg to 50 ng of input DNA. Using assembly-based metagenomic analysis, we demonstrate that DNA input level has a significant impact on community structure due to overrepresentation of low-GC genomic regions following library amplification. In our system, these differences were largely superseded by variations between biological replicates, but our results advocate verifying the influence of library amplification on a case-by-case basis. Overall, this study provides recommendations for quality filtering and de-replication prior to analysis, as well as a practical framework to address the issue of low biomass or biomass heterogeneity in longitudinal metagenomic surveys.

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Section: Effects Of Library Preparation On Metagenomic Library Qualitymentioning

confidence: 99%

Section: Effects Of Library Preparation On Metagenomic Library Qualitymentioning

confidence: 99%

The effects of variable sample biomass on comparative metagenomics

Chafee

Maignien

Simmons

2015

Environmental Microbiology

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“…Exome sequencing studies at moderate (approximately 100X) depth rely on read position to identify potential PCR duplicates [17], but amplicon-based (molecular inversion probes [18], RainDrop Digital PCR (RainDance Technologies, Billerica, MA, USA), TruSeq Custom Amplicon (Illumina, San Diego, CA, USA)) methods commonly used for targeted sequencing have reads with the same start and stop positions. Hybridization-based methods, when sequenced deeply, can result in reads that are not PCR duplicates but have the same start stop locations by chance [19]. PCR-free methods are also available, but typically require higher amounts of DNA input (1 to 2 ug), limiting their use in cancer studies.…”

Section: Introductionmentioning

confidence: 99%

Biased estimates of clonal evolution and subclonal heterogeneity can arise from PCR duplicates in deep sequencing experiments

et al. 2014

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Accurate allele frequencies are important for measuring subclonal heterogeneity and clonal evolution. Deep-targeted sequencing data can contain PCR duplicates, inflating perceived read depth. Here we adapted the Illumina TruSeq Custom Amplicon kit to include single molecule tagging (SMT) and show that SMT-identified duplicates arise from PCR. We demonstrate that retention of PCR duplicate reads can imply clonal evolution when none exists, while their removal effectively controls the false positive rate. Additionally, PCR duplicates alter estimates of subclonal heterogeneity in tumor samples. Our method simplifies PCR duplicate identification and emphasizes their removal in studies of tumor heterogeneity and clonal evolution.

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“…T200·2 target‐capture deep‐sequencing data were aligned to human hg19 using BWA and duplicate reads were removed using Picard . A proprietary custom analysis pipeline classified somatic variants on the basis of variant allele frequencies in tumour and matched normal tissue. Functional consequences of somatic variants were assessed by comparison with the dbSNP, COSMIC and TCGA databases and then annotated using VEP, Annovar and CanDrA …”

Section: Methodsmentioning

confidence: 99%

Spitzoid melanoma with histopathological features of ALK gene rearrangement exhibiting ALK copy number gain: a novel mechanism of ALK activation in spitzoid neoplasia

et al. 2018

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Spitzoid neoplasms pose diagnostic difficulties because their morphology is not consistently predictive of their biological potential. Recent advances in the molecular characterization of these tumours provides a framework by which they can now begin to be categorized. In particular, spitzoid lesions with ALK rearrangement have been specifically associated with a characteristic plexiform growth pattern of intersecting fascicles of amelanotic spindled melanocytes. We report the case of an 87-year-old man with a 3-cm nodule on his mid-upper back comprised of an intradermal proliferation of fusiform amelanotic melanocytes arranged in intersecting fascicles with occasional peritumoral clefts. Immunohistochemical studies demonstrated diffuse, strong expression of SOX10 and S100 by the tumour cells and diffuse, weak-to-moderate cytoplasmic positivity for anaplastic lymphoma kinase (ALK), suggestive of ALK rearrangement. Fluorescence in situ hybridization revealed no ALK rearrangements but instead revealed at least three intact ALK signals in 36% of the tumour cells, confirming ALK copy number gain. To our knowledge, this is the first reported case of a plexiform spitzoid neoplasm exhibiting ALK copy number gain instead of ALK rearrangement. This case suggests that ALK copy number gain is a novel mechanism of ALK activation but with the same characteristic histopathological growth pattern seen among ALK-rearranged spitzoid neoplasms.

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Bias from removing read duplication in ultra-deep sequencing experiments

Abstract: A Python implementation is freely available at https://bitbucket.org/wanding/duprecover/overview CONTACT: : wzhou1@mdanderson.org, kchen3@mdanderson.org Supplementary information: Supplementary data are available at Bioinformatics online.

Cited by 39 publications

References 16 publications

The effects of variable sample biomass on comparative metagenomics

The effects of variable sample biomass on comparative metagenomics

Biased estimates of clonal evolution and subclonal heterogeneity can arise from PCR duplicates in deep sequencing experiments

Spitzoid melanoma with histopathological features of ALK gene rearrangement exhibiting ALK copy number gain: a novel mechanism of ALK activation in spitzoid neoplasia

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