Introduction

Bwakit is a self-consistent installation-free package of scripts and precompiled binaries, providing an end-to-end solution to read mapping. In addition to the basic mapping functionality implemented in bwa, bwakit is able to generate proper human reference genome and to take advantage of ALT contigs, if present, to improve read mapping and to perform HLA typing for high-coverage human data. It can remap name- or coordinate-sorted BAM with read group and barcode information retained. Bwakit also optionally trims adapters (via trimadap), marks duplicates (via samblaster) and sorts the final alignment (via samtools).

Bwakit has two entry scripts: run-gen-ref which downloads and generates human reference genomes, and run-bwamem which prints mapping command lines on the standard output that can be piped to sh to execute. The two scripts will call other programs or use data in bwa.kit. The following shows an example about how to use bwakit:

# Download the bwa-0.7.11 binary package (download link may change)
wget -O- http://sourceforge.net/projects/bio-bwa/files/bwakit/bwakit-0.7.12_x64-linux.tar.bz2/download \
  | gzip -dc | tar xf -
# Generate the GRCh38+ALT+decoy+HLA and create the BWA index
bwa.kit/run-gen-ref hs38DH   # download GRCh38 and write hs38DH.fa
bwa.kit/bwa index hs38DH.fa  # create BWA index
# mapping
bwa.kit/run-bwamem -o out -H hs38DH.fa read1.fq read2.fq | sh

The last mapping command line will generate the following files:

• out.aln.bam: unsorted alignments with ALT-aware mapping quality. In this file, one read may be placed on multiple overlapping ALT contigs at the same time even if the read is mapped better to some contigs than others. This makes it possible to analyze each contig independent of others.

• out.hla.top: best genotypes for HLA-A, -B, -C, -DQA1, -DQB1 and -DRB1 genes.

• out.hla.all: other possible genotypes on the six HLA genes.

• out.log.*: bwa-mem, samblaster and HLA typing log files.

Bwakit can be downloaded here. It is only available to x86_64-linux. The scripts in the package are available in the bwa/bwakit directory. Packaging is done manually for now.

Limitations

• HLA typing only works for high-coverage human data. The typing accuracy can still be improved. We encourage researchers to develop better HLA typing tools based on the intermediate output of bwakit (for each HLA gene included in the index, bwakit writes all reads matching it in a separate file).

• Duplicate marking only works when all reads from a single paired-end library are provided as the input. This limitation is the necessary tradeoff of fast MarkDuplicate provided by samblaster.

• The adapter trimmer is chosen as it is fast, pipe friendly and does not discard reads. However, it is conservative and suboptimal. If this is a concern, it is recommended to preprocess input reads with a more sophisticated adapter trimmer. We also hope existing trimmers can be modified to operate on an interleaved FASTQ stream. We will replace trimadap once a better trimmer meets our needs.

• Bwakit can be memory demanding depends on the functionality invoked. For 30X human data, bwa-mem takes about 11GB RAM with 32 threads, samblaster uses close to 10GB and BAM shuffling (if the input is sorted BAM) uses several GB. In the current setting, sorting uses about 10GB.

Package Contents

bwa.kit
|-- README.md                  This README file.
|-- run-bwamem                 *Entry script* for the entire mapping pipeline.
|-- bwa                        *BWA binary*
|-- k8                         Interpretor for *.js scripts.
|-- bwa-postalt.js             Post-process alignments to ALT contigs/decoys/HLA genes.
|-- htsbox                     Used by run-bwamem for shuffling BAMs and BAM=>FASTQ.
|-- samblaster                 MarkDuplicates for reads from the same library. v0.1.20
|-- samtools                   SAMtools for sorting and SAM=>BAM conversion. v1.1
|-- seqtk                      For FASTQ manipulation.
|-- trimadap                   Trim Illumina PE sequencing adapters.
|
|-- run-gen-ref                *Entry script* for generating human reference genomes.
|-- resource-GRCh38            Resources for generating GRCh38
|   |-- hs38DH-extra.fa        Decoy and HLA gene sequences. Used by run-gen-ref.
|   `-- hs38DH.fa.alt          ALT-to-GRCh38 alignment. Used by run-gen-ref.
|
|-- run-HLA                    HLA typing for sequences extracted by bwa-postalt.js.
|-- typeHLA.sh                 Type one HLA-gene. Called by run-HLA.
|-- typeHLA.js                 HLA typing from exon-to-contig alignment. Used by typeHLA.sh.
|-- typeHLA-selctg.js          Select contigs overlapping HLA exons. Used by typeHLA.sh.
|-- fermi2.pl                  Fermi2 wrapper. Used by typeHLA.sh for de novo assembly.
|-- fermi2                     Fermi2 binary. Used by fermi2.pl.
|-- ropebwt2                   RopeBWT2 binary. Used by fermi2.pl.
|-- resource-human-HLA         Resources for HLA typing
|   |-- HLA-ALT-exons.bed      Exonic regions of HLA ALT contigs. Used by typeHLA.sh.
|   |-- HLA-CDS.fa             CDS of HLA-{A,B,C,DQA1,DQB1,DRB1} genes from IMGT/HLA-3.18.0.
|   |-- HLA-ALT-type.txt       HLA types for each HLA ALT contig. Not used.
|   `-- HLA-ALT-idx            BWA indices of each HLA ALT contig. Used by typeHLA.sh
|       `-- (...)
|
`-- doc                        BWA documentations
    |-- bwa.1                  Manpage
    |-- NEWS.md                Release Notes
    |-- README.md              GitHub README page
    `-- README-alt.md          Documentation for ALT mapping