amplimap documentation, version 0.4

An amplicon mapping and analysis pipeline

amplimap is a pipeline to process and analyse short-read sequencing data from smMIP-panels or PCR-based amplicons. It was designed to support both germline variant calling as well as quantification of variant allele frequencies using ultra-high-coverage pileups with and without UMIs.

amplimap takes fastq or bam files generated from an Illumina sequencer (tested with MiSeq, HiSeq, and NextSeq), assigns each read pair to a probe/amplicon, trims and aligns the reads and then generates a set of basic statistics. After that, two different types of analyses can be performed:

1. Germline variant calling using Platypus/GATK and annotation with Annovar, to generate an annotated table of potentially pathogenic variants.
2. Per-basepair “pileup” of reads to generate nucleotide counts of each base at each target basepair and each sample, for analysis of allele frequencies.

Built on top of Snakemake and Python 3, amplimap is entirely automated and can be run on a single machine as well as on a HPC cluster (eg. LSF, SGE).

Contents

• Installation
  • Setup
  • Requirements
• Quickstart
  • Pipeline overview
• Input and the working directory
  • Input overview
  • Input files
• Running amplimap
  • bams (read parsing and alignment)
  • variants and variants_umi (germline variant calling/annotation)
  • coverages (target coverage tables)
  • pileups (allele counts and allele frequencies)
  • variants_low_frequency (low-frequency/somatic variant calling)
  • Multiple targets
  • Running on a cluster
• Output: the analysis directory
  • Target-specific output
  • Additional output
• Configuration
  • Default configuration
  • Local configuration
  • Common configuration changes
  • Support for modules
  • All configuration options
• Tutorials
• Advanced usage
  • Ignoring UMI groups
  • Running on capture-based data
  • Simulating variation
  • Merging runs
  • Additional Notes
• Extended installation guide for amplimap
  • 1. Install bioconda
  • 2. Setup amplimap enviroment
  • 3. Activate amplimap
  • 4. Set up your reference genome and indices
  • 5. Update amplimap configuration
  • 6. Run amplimap!
• Code documentation
  • Common
  • File reader
  • Read parser
  • Alignment stats
  • Coverage
  • Variants
  • Pileup
  • Others
• References
  • Software required by amplimap
  • Recommended additional software

Citation and License

Licensed under the Apache License, version 2.0. Copyright 2018 Nils Koelling. When you use amplimap, please cite it in your work. For example:

Koelling, Nils. “amplimap: amplicon mapping and analysis pipeline (version 0.4)”. https://github.com/koelling/amplimap/