Updated README for ALT mapping
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Heng Li
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README-alt.md
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README-alt.md
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@ -1,57 +1,63 @@
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## Getting Started
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Since version 0.7.11, BWA-MEM supports read mapping against a reference genome
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with long alternative haplotypes present in separate ALT contigs. To use the
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ALT-aware mode, users need to provide pairwise ALT-to-reference alignment in the
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SAM format and rename the file to "*idxbase*.alt". For GRCh38, this alignment
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is available from the [binary package of BWA][res].
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```sh
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# Generate the GRCh38+ALT+decoy+HLA and create the BWA index
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# Download the bwa-0.7.11 binary package
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wget -O- http://sourceforge.net/projects/bio-bwa/files/bwakit-0.7.11_x64-linux.tar.bz2/download \
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| gzip -dc | tar xf -
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bwa.kit/run-gen-hs38d6 # download GRCh38 and write hs38d6.fa
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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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# mapping
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bwa.kit/run-bwamem hs38d6.fa read1.fq read2.fq | sh
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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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```
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In the final alignment, a read may be placed on the [primary assembly][grcdef]
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and multiple overlapping ALT contigs at the same time (on multiple SAM lines).
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Mapping quality (mapQ) is properly adjusted by the postprocessing script
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`bwa-postalt.js` using the ALT-to-reference alignment `hs38a.fa.alt`. For
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details, see the [Methods section](#methods).
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This will generate the following files:
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* `out.aln.sam.gz`: unsorted alignments with ALT-aware mapping quality. In this
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file, one read may be placed on multiple overlapping ALT contigs at the same
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time even if the read is mapped better to some contigs than others. This makes
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it possible to analyze each contig independent of others.
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* `out.hla.top`: best genotypes for HLA-A, -B, -C, -DQA1, -DQB1 and -DRB1 genes.
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* `out.hla.all`: other possible genotypes on the six HLA genes.
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* `out.log.*`: bwa-mem, samblaster and HLA typing log files.
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Note that `run-bwamem` only prints command lines but doesn't execute them. It
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is advised to have a look at the command lines before passing them to `sh` for
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actual execution.
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## Background
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GRCh38 consists of several components: chromosomal assembly, unlocalized contigs
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(chromosome known but location unknown), unplaced contigs (chromosome unknown)
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and ALT contigs (long clustered variations). The combination of the first three
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components is called the *primary assembly*. You can find the more exact
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definitions from the [GRC website][grcdef].
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components is called the *primary assembly*. It is recommended to use the
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complete primary assembly for all analyses. Using ALT contigs in read mapping is
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tricky.
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GRCh38 ALT contigs are totaled 109Mb in length, spanning 60Mbp genomic regions.
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However, sequences that are highly diverged from the primary assembly only
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contribute a few million bp. Most subsequences of ALT contigs are nearly
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GRCh38 ALT contigs are totaled 109Mb in length, spanning 60Mbp of the primary
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assembly. However, sequences that are highly diverged from the primary assembly
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only contribute a few million bp. Most subsequences of ALT contigs are nearly
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identical to the primary assembly. If we align sequence reads to GRCh38+ALT
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treating ALT equal to the primary assembly, we will get many reads with zero
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mapping quality and lose variants on them. It is crucial to make the mapper
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aware of ALTs.
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blindly, we will get many additional reads with zero mapping quality and miss
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variants on them. It is crucial to make mappers aware of ALTs.
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BWA-MEM is designed to minimize the interference of ALT contigs such that on the
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primary assembly, the ALT-aware alignment is highly similar to the alignment
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without using ALT contigs in the index. This design choice makes it almost
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always safe to map reads to GRCh38+ALT. Although we don't know yet how much
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variations on ALT contigs contribute to phenotypes, we would not get the answer
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without mapping large cohorts to these extra sequences. We hope our current
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implementation encourages researchers to use ALT contigs soon and often.
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BWA-MEM is ALT-aware. It essentially computes mapping quality across the
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non-redundant content of the primary assembly plus the ALT contigs and is free
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of the problem above.
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## Methods
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### Sequence alignment
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As of now, ALT mapping is done in two separate steps: BWA-MEM mapping and
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postprocessing.
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postprocessing. The `bwa.kit/run-bwamem` script performs the two steps when ALT
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contigs are present. The following picture shows an example about how BWA-MEM
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infers mapping quality and reports alignment after step 2:
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#### Step 1: BWA-MEM mapping
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@ -65,11 +71,11 @@ alignments and assigns mapQ following these two rules:
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2. If there are no non-ALT hits, the best ALT hit is outputted as the primary
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alignment. If there are both ALT and non-ALT hits, non-ALT hits will be
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primary. ALT hits are reported as supplementary alignments (flag 0x800) only
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if they are better than all overlapping non-ALT hits.
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primary and ALT hits be supplementary (SAM flag 0x800) if ALT hits are better
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than the best overlapping non-ALT hits.
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In theory, non-ALT alignments from step 1 should be identical to alignments
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against a reference genome with ALT contigs. In practice, the two types of
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against the reference genome with ALT contigs. In practice, the two types of
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alignments may differ in rare cases due to seeding heuristics. When an ALT hit
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is significantly better than non-ALT hits, BWA-MEM may miss seeds on the
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non-ALT hits.
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@ -102,32 +108,32 @@ 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 `hs38d4-extra.fa` from the [BWA resource bundle
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for GRCh38][res].
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The sequences are included in `hs38d6-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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`hs38d4-extra.fa`. These genomic sequences were acquired from [IMGT/HLA][hladb],
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version 3.18.0. Script `bwa-postalt.js` also helps to genotype HLA genes, though
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not to high resolution for now.
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`hs38d6-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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### More on HLA typing
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### HLA typing
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It is [well known][hlalink] that HLA genes are associated with many autoimmunity
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infectious diseases and drug responses. However, many HLA alleles are highly
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diverged from the reference genome. If we map whole-genome shotgun (WGS) reads
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to the reference only, many allele-informative will get lost. As a result, the
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vast majority of WGS projects have ignored these important genes.
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HLA genes are known to be associated with many autoimmune diseases, infectious
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diseases and drug responses. They are among the most important genes but are
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rarely studied by WGS projects due to the high sequence divergence between
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HLA genes and the reference genome in these regions.
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We recommend to include the genomic regions of classical HLA genes in the BWA
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index. This way we will be able to get a more complete collection of reads
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mapped to HLA. We can then isolate these reads with little computational cost
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and type HLA genes with another program, such as [Warren et al (2012)][hla4],
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[Liu et al (2013)][hla2], [Bai et al (2014)][hla3], [Dilthey et al (2014)][hla1]
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or others from [this list][hlatools].
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### Evaluating ALT Mapping
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(Coming soon...)
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By including the HLA gene regions in the reference assembly as ALT contigs, we
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are able to effectively identify reads coming from these genes. We also provide
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a pipeline, which is included in the [BWA binary package][res], to type the
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several classic HLA genes. The pipeline is conceptually simple. It de novo
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assembles sequence reads mapped to each gene, aligns exon sequences of each
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allele to the assembled contigs and then finds the pairs of alleles that best
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explain the contigs. In practice, however, the completeness of IMGT/HLA and
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copy-number changes related to these genes are not so straightforward to
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resolve. HLA typing may not always be successful. Users may also consider to use
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other programs for typing such as [Warren et al (2012)][hla4], [Liu et al
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(2013)][hla2], [Bai et al (2014)][hla3] and [Dilthey et al (2014)][hla1], though
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most of them are distributed under restrictive licenses.
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## Problems and Future Development
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\f1\b \cf8 ATCgcCAgC
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\f0\b0 \cf0 >
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||||
<string>float</string>
|
||||
<string>18</string>
|
||||
</array>
|
||||
<key>NSPaperSize</key>
|
||||
<array>
|
||||
<string>size</string>
|
||||
<string>{612, 792}</string>
|
||||
</array>
|
||||
<key>NSPrintReverseOrientation</key>
|
||||
<array>
|
||||
<string>int</string>
|
||||
<string>0</string>
|
||||
</array>
|
||||
<key>NSRightMargin</key>
|
||||
<array>
|
||||
<string>float</string>
|
||||
<string>18</string>
|
||||
</array>
|
||||
<key>NSTopMargin</key>
|
||||
<array>
|
||||
<string>float</string>
|
||||
<string>18</string>
|
||||
</array>
|
||||
</dict>
|
||||
<key>PrintOnePage</key>
|
||||
<false/>
|
||||
<key>ReadOnly</key>
|
||||
<string>NO</string>
|
||||
<key>RowAlign</key>
|
||||
<integer>1</integer>
|
||||
<key>RowSpacing</key>
|
||||
<real>36</real>
|
||||
<key>SheetTitle</key>
|
||||
<string>Canvas 1</string>
|
||||
<key>SmartAlignmentGuidesActive</key>
|
||||
<string>YES</string>
|
||||
<key>SmartDistanceGuidesActive</key>
|
||||
<string>YES</string>
|
||||
<key>UniqueID</key>
|
||||
<integer>1</integer>
|
||||
<key>UseEntirePage</key>
|
||||
<false/>
|
||||
<key>VPages</key>
|
||||
<integer>1</integer>
|
||||
<key>WindowInfo</key>
|
||||
<dict>
|
||||
<key>CurrentSheet</key>
|
||||
<integer>0</integer>
|
||||
<key>ExpandedCanvases</key>
|
||||
<array>
|
||||
<dict>
|
||||
<key>name</key>
|
||||
<string>Canvas 1</string>
|
||||
</dict>
|
||||
</array>
|
||||
<key>Frame</key>
|
||||
<string>{{367, 6}, {710, 872}}</string>
|
||||
<key>ListView</key>
|
||||
<true/>
|
||||
<key>OutlineWidth</key>
|
||||
<integer>142</integer>
|
||||
<key>RightSidebar</key>
|
||||
<false/>
|
||||
<key>ShowRuler</key>
|
||||
<true/>
|
||||
<key>Sidebar</key>
|
||||
<true/>
|
||||
<key>SidebarWidth</key>
|
||||
<integer>120</integer>
|
||||
<key>VisibleRegion</key>
|
||||
<string>{{0, 0}, {575, 733}}</string>
|
||||
<key>Zoom</key>
|
||||
<real>1</real>
|
||||
<key>ZoomValues</key>
|
||||
<array>
|
||||
<array>
|
||||
<string>Canvas 1</string>
|
||||
<real>1</real>
|
||||
<real>1</real>
|
||||
</array>
|
||||
</array>
|
||||
</dict>
|
||||
</dict>
|
||||
</plist>
|
||||
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|
After Width: | Height: | Size: 45 KiB |
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Reference in New Issue