improved doc and don't type HLA by default
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Heng Li
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11
NEWS.md
11
NEWS.md
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@ -1,17 +1,18 @@
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Release 0.7.11 (XX November, 2014)
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-----------------------------------
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Release 0.7.11 (XX December, 2014)
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----------------------------------
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A major change to BWA-MEM is the support of mapping to ALT contigs in addition
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to the primary assembly. Part of the ALT mapping strategy is implemented in
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BWA-MEM and the rest in a postprocessing script for now. Due to the extra
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layer of complexity on generating the reference genome and on the two-step
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mapping, we start to provide a wrapper script and precompiled binaries since
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this release. Please check README-alt.md for details.
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this release. The package may be more convenient to some specific use cases.
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For general uses, the single BWA binary still works like the old way.
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Another major addition to BWA-MEM is HLA typing, which made possible with the
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new ALT mapping strategy. Necessary data and programs are included in the
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binary release. The wrapper script also performs HLA typing when HLA genes are
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also included in the reference genome as additional ALT contigs.
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included in the reference genome as additional ALT contigs.
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Other notable changes to BWA-MEM:
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@ -44,7 +45,7 @@ Other notable changes to BWA-MEM:
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writing SAM. This saves significant wall-clock time when reading from
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or writing to a slow Unix pipe.
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(0.7.11: XX November 2014, rXXX)
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(0.7.11: XX December 2014, r10XX)
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@ -5,10 +5,10 @@
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wget -O- http://sourceforge.net/projects/bio-bwa/files/bwakit/bwakit-0.7.11_x64-linux.tar.bz2/download \
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| gzip -dc | tar xf -
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# Generate the GRCh38+ALT+decoy+HLA and create the BWA index
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bwa.kit/run-gen-ref hs38d6 # download GRCh38 and write hs38d6.fa
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bwa.kit/bwa index hs38d6.fa # create BWA index
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bwa.kit/run-gen-ref hs38D1 # download GRCh38 and write hs38D1.fa
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bwa.kit/bwa index hs38D1.fa # create BWA index
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# mapping
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bwa.kit/run-bwamem -o out hs38d6.fa read1.fq read2.fq | sh # skip "|sh" to show command lines
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bwa.kit/run-bwamem -o out -H hs38D1.fa read1.fq read2.fq | sh # skip "|sh" to show command lines
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```
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This generates `out.aln.bam` as the final alignment, `out.hla.top` for best HLA
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@ -94,11 +94,11 @@ CHM1 short reads and present also in NA12878. You can try [BLAT][blat] or
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For a more complete reference genome, we compiled a new set of decoy sequences
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from GenBank clones and the de novo assembly of 254 public [SGDP][sgdp] samples.
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The sequences are included in `hs38d6-extra.fa` from the [BWA binary
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The sequences are included in `hs38D1-extra.fa` from the [BWA binary
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package][res].
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In addition to decoy, we also put multiple alleles of HLA genes in
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`hs38d6-extra.fa`. These genomic sequences were acquired from [IMGT/HLA][hladb],
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`hs38D1-extra.fa`. These genomic sequences were acquired from [IMGT/HLA][hladb],
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version 3.18.0 and are used to collect reads sequenced from these genes.
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### HLA typing
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@ -125,26 +125,26 @@ most of them are distributed under restrictive licenses.
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To check whether GRCh38 is better than GRCh37, we mapped the CHM1 and NA12878
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unitigs to GRCh37 primary (hs37), GRCh38 primary (hs38) and GRCh38+ALT+decoy
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(hs38d6), and called small variants from the alignment. CHM1 is haploid.
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(hs38D1), and called small variants from the alignment. CHM1 is haploid.
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Ideally, heterozygous calls are false positives (FP). NA12878 is diploid. The
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true positive (TP) heterozygous calls from NA12878 are approximately equal
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to the difference between NA12878 and CHM1 heterozygous calls. A better assembly
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should yield higher TP and lower FP. The following table shows the numbers for
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these assemblies:
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|Assembly|hs37 |hs38 |hs38d6|CHM1_1.1| huref|
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|Assembly|hs37 |hs38 |hs38D1|CHM1_1.1| huref|
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|:------:|------:|------:|------:|------:|------:|
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|FP | 255706| 168068| 142516|307172 | 575634|
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|TP |2142260|2163113|2150844|2167235|2137053|
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With this measurement, hs38 is clearly better than hs37. Genome hs38d6 reduces
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With this measurement, hs38 is clearly better than hs37. Genome hs38D1 reduces
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FP by ~25k but also reduces TP by ~12k. We manually inspected variants called
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from hs38 only and found the majority of them are associated with excessive read
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depth, clustered variants or weak alignment. We believe most hs38-only calls are
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problematic. In addition, if we compare two NA12878 replicates from HiSeq X10
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with nearly identical library construction, the difference is ~140k, an order
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of magnitude higher than the difference between hs38 and hs38d6. ALT contigs,
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decoy and HLA genes in hs38d6 improve variant calling and enable the analyses of
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of magnitude higher than the difference between hs38 and hs38D1. ALT contigs,
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decoy and HLA genes in hs38D1 improve variant calling and enable the analyses of
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ALT contigs and HLA typing at little cost.
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## Problems and Future Development
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@ -21,10 +21,10 @@ how to use bwakit:
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wget -O- http://sourceforge.net/projects/bio-bwa/files/bwakit/bwakit-0.7.11_x64-linux.tar.bz2/download \
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| gzip -dc | tar xf -
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# Generate the GRCh38+ALT+decoy+HLA and create the BWA index
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bwa.kit/run-gen-ref hs38d6 # download GRCh38 and write hs38d6.fa
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bwa.kit/bwa index hs38d6.fa # create BWA index
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bwa.kit/run-gen-ref hs38D1 # download GRCh38 and write hs38D1.fa
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bwa.kit/bwa index hs38D1.fa # create BWA index
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# mapping
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bwa.kit/run-bwamem -o out hs38d6.fa read1.fq read2.fq | sh
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bwa.kit/run-bwamem -o out -H hs38D1.fa read1.fq read2.fq | sh
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```
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The last mapping command line will generate the following files:
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@ -44,7 +44,31 @@ Bwakit can be [downloaded here][res]. It is only available to x86_64-linux. The
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scripts in the package are available in the [bwa/bwakit][kit] directory.
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Packaging is done manually for now.
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## Contents
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## Limitations
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* HLA typing only works for high-coverage human data. The typing accuracy can
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still be improved. We encourage researchers to develop better HLA typing tools
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based on the intermediate output of bwakit (for each HLA gene included in the
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index, bwakit writes all reads matching it in a separate file).
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* Duplicate marking only works when all reads from a single paired-end library
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are provided as the input. This limitation is the necessary tradeoff of fast
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MarkDuplicate provided by samblaster.
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* The adapter trimmer is chosen as it is fast, pipe friendly and does not
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discard reads. However, it is conservative and suboptimal. If this is a
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concern, it is recommended to preprocess input reads with a more sophisticated
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adapter trimmer. We also hope existing trimmers can be modified to operate on
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an interleaved FASTQ stream. We will replace trimadap once a better trimmer
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meets our needs.
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* Bwakit can be memory demanding depends on the functionality invoked. For 30X
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human data, bwa-mem takes about 6GB RAM, samblaster uses close to 10GB and BAM
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shuffling (if the input is sorted BAM) uses several GB. In the current
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setting, sorting uses about 10GB.
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## Package Contents
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```
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bwa.kit
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|-- README.md This README file.
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@ -18,29 +18,38 @@ Options: -o STR prefix for output files [inferred from
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ont2d: Oxford Nanopore reads (~10kb query, higher error rate)
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-t INT number of threads [1]
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-H apply HLA typing
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-a trim HiSeq2000/2500 PE resequencing adapters (via trimadap)
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-d mark duplicate (via samblaster)
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-S for SAM/BAM input, don\'t shuffle
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-s sort the output alignment (requring more RAM)
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-H skip HLA typing
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-S for BAM input, don\'t shuffle
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-s sort the output alignment (via samtools; requring more RAM)
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-k keep temporary files generated by typeHLA
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Examples:
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* Map paired-end reads to GRCh38+ALT+decoy+HLA and perform HLA typing:
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run-bwamem -o prefix -t8 -R"@RG\tID:foo\tSM:bar" hs38d6.fa read1.fq.gz read2.fq.gz
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run-bwamem -o prefix -t8 -HR"@RG\tID:foo\tSM:bar" hs38D1.fa read1.fq.gz read2.fq.gz
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Note: HLA typing is only effective for high-coverage data. The typing accuracy varies
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with the quality of input. It is only intended for research purpose, not for diagnostic.
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* Remap coordinate-sorted BAM, transfer read groups tags, trim Illumina PE adapters and
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sort the output. The BAM may contain single-end or paired-end reads, or a mixture of
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the two types. Specifying -R stops read group transfer.
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run-bwamem -sao prefix hs38d6.fa old-srt.bam
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run-bwamem -sao prefix hs38D1.fa old-srt.bam
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* Remap name-grouped BAM and mark duplicates. Note that in this case, all reads from
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a single library should be aligned at the same time. Paired-end only.
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Note: the adaptor trimmer included in bwa.kit is chosen because it fits the current
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mapping pipeline better. It is conservative and suboptimal. A more sophisticated
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trimmer is recommended if this becomes a concern.
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run-bwamem -Sdo prefix hs38d6.fa old-unsrt.bam
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* Remap name-grouped BAM and mark duplicates:
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run-bwamem -Sdo prefix hs38D1.fa old-unsrt.bam
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Note: streamed duplicate marking requires all reads from a single paired-end library
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to be aligned at the same time.
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Output files:
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@ -156,7 +165,7 @@ if (-f "$ARGV[0].alt") {
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my $t_sort = $opts{t} < 4? $opts{t} : 4;
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$cmd .= defined($opts{s})? " | $root/samtools sort -@ $t_sort -m1G - $prefix.aln;\n" : " | $root/samtools view -1 - > $prefix.aln.bam;\n";
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if ($has_hla && !defined($opts{H}) && (!defined($opts{x}) || $opts{x} eq 'intractg')) {
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if ($has_hla && defined($opts{H}) && (!defined($opts{x}) || $opts{x} eq 'intractg')) {
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$cmd .= "$root/run-HLA ". (defined($opts{x}) && $opts{x} eq 'intractg'? "-A " : "") . "$prefix.hla > $prefix.hla.top 2> $prefix.log.hla;\n";
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$cmd .= "cat $prefix.hla.HLA*.gt | grep ^GT | cut -f2- > $prefix.hla.all;\n";
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$cmd .= "rm -f $prefix.hla.HLA*;\n" unless defined($opts{k});
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