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Examining Sources of Error in PCR by Single-Molecule Sequencing

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Examining Sources of Error in PCR by Single-Molecule Sequencing

Vladimir Potapov and Jennifer L. Ong

Description

This repository provides a set of scripts for a PacBio single-molecule sequencing assay used to comprehensively catalog the different types of errors introduced during PCR. The full details of the method are available in the following publication:

Potapov V. & Ong JL. Examining Sources of Error in PCR by Single-Molecule Sequencing. PLOS ONE. 2016. doi:10.1371/journal.pone.0169774. (View article)

Usage

The analysis workflow consists of several consecutive steps as outlined below. The correspondng scripts can be found in scripts/ directory.

  1. Generating strand-specific consensus sequences (ccs2-consensus.sh).

  2. Mapping consensus sequences to a reference sequence and extracting mutations (ccs2-mutation.sh).

  3. Filtering and summarizing mutations (ccs2-chimeric.sh,ccs2-summary.sh)

  4. Additional scripts to analyze template switching and PCR-mediated recombination can be found in extra/ directory.

The included workflow provides detailed instructions for data analysis conducted in the original publication (1).

Requirements

  • SMRT Link. Scripts rely on bax2bam, ccs and blasr command-line utilities, which are developed by Pacific Biosciences, Inc. and distributed as part of the SMRT Link software.
  • SAMtools. Manipulating BAM files.
  • BWA. Detecting chimeric reads.
  • P7ZIP. Compressing output files to minimize disk usage.
  • The R Project for Statistical Computing. Extracting and tabulating data.

Citations

  1. Potapov V. & Ong JL. Examining Sources of Error in PCR by Single-Molecule Sequencing. PLOS ONE. 2016. doi:10.1371/journal.pone.0169774. (View article)

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Examining Sources of Error in PCR by Single-Molecule Sequencing

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