An Improved Sequencing-Based Bioinformatics Pipeline to Track the Distribution and Clonal Architecture of Proviral Integration Sites

Frontiers in Microbiology
N RosewickA Van den Broeke

Abstract

The combined application of linear amplification-mediated PCR (LAM-PCR) protocols with next-generation sequencing (NGS) has had a large impact on our understanding of retroviral pathogenesis. Previously, considerable effort has been expended to optimize NGS methods to explore the genome-wide distribution of proviral integration sites and the clonal architecture of clinically important retroviruses like human T-cell leukemia virus type-1 (HTLV-1). Once sequencing data are generated, the application of rigorous bioinformatics analysis is central to the biological interpretation of the data. To better exploit the potential information available through these methods, we developed an optimized bioinformatics pipeline to analyze NGS clonality datasets. We found that short-read aligners, specifically designed to manage NGS datasets, provide increased speed, significantly reducing processing time and decreasing the computational burden. This is achieved while also accounting for sequencing base quality. We demonstrate the utility of an additional trimming step in the workflow, which adjusts for the number of reads supporting each insertion site. In addition, we developed a recall procedure to reduce bias associated with proviral integ...Continue Reading

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Citations

Apr 13, 2021·The Journal of Infectious Diseases·Hiroo KatsuyaYorifumi Satou

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Datasets Mentioned

BETA
KT122858.1
or
J02029
sequence

Methods Mentioned

BETA
PCR

Software Mentioned

R scripts
custom
seqtk
cutadapt
fastq
bowtie2
BLAT
fasta
to
SAM

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