fast-bwa/README-alt.md

Getting Started

Since version 0.7.11, BWA-MEM supports read mapping against a reference genome with long alternative haplotypes present in separate ALT contigs. To use the ALT-aware mode, users need to provide pairwise ALT-to-reference alignment in the SAM format and rename the file to ""idxbase.alt". For GRCh38, this alignment is available from the BWA resource bundle for GRCh38.

Option 1: Mapping to the official GRCh38 with ALT contigs

Construct the index:

wget ftp://ftp.ncbi.nlm.nih.gov/genbank/genomes/Eukaryotes/vertebrates_mammals/Homo_sapiens/GRCh38/seqs_for_alignment_pipelines/GCA_000001405.15_GRCh38_full_analysis_set.fna.gz
gzip -d GCA_000001405.15_GRCh38_full_analysis_set.fna.gz
mv GCA_000001405.15_GRCh38_full_analysis_set.fna hs38a.fa
bwa index hs38a.fa
cp bwa-hs38-res/hs38d4.fa.alt hs38a.fa.alt

Perform mapping:

bwa mem hs38a.fa read1.fq read2.fq \
  | samblaster \
  | bwa-hs38-res/k8-linux bwa-postalt.js hs38a.fa.alt \
  | samtools view -bS - > aln.unsrt.bam

For short reads, the postprocessing script bwa-postalt.js runs at about the same speed as BAM compression.

Option 2: Mapping to the collection of GRCh38, decoy and HLA genes

Construct the index:

cat hs38a.fa bwa-hs38-res/hs38d4-extra.fa > hs38d4.fa
bwa index hs38d4.fa
cp bwa-hs38-res/hs38d4.fa.alt .

Perform mapping:

bwa mem hs38d4.fa read1.fq read2.fq \
  | samblaster \
  | bwa-hs38-res/k8-linux bwa-postalt.js -p postinfo hs38d4.fa.alt \
  | samtools view -bS - > aln.unsrt.bam

This command line generates postinfo.ctw which loosely evaluates the presence of an ALT contig with an empirical score at the last column.

If you are not interested in the way BWA-MEM performs ALT mapping, you can skip the rest of this documentation.

Background

GRCh38 ALT contigs are totaled 109Mb in length, spanning 60Mbp genomic regions. However, sequences that are highly diverged from the primary assembly only contribute a few million bp. Most subsequences of ALT contigs are highly similar or identical to the primary assembly. If we align sequence reads to GRCh38+ALT treating ALT equal to the primary assembly, we will get many reads with zero mapping quality and lose variants on them. It is crucial to make the mapper aware of ALTs.

BWA-MEM is designed to minimize the interference of ALT contigs such that on the primary assembly, the ALT-aware alignment is highly similar to the alignment without using ALT contigs in the index. This design choice makes it almost always safe to map reads to GRCh38+ALT. Although we don't know yet how much variations on ALT contigs contribute to phenotypes, we would not get the answer without mapping large cohorts to these extra sequences. We hope our current implementation encourages researchers to use ALT contigs soon and often.

Methods

Sequence alignment

As of now, ALT mapping is done in two separate steps: BWA-MEM mapping and postprocessing.

Step 1: BWA-MEM mapping

At this step, BWA-MEM reads the ALT contig names from "idxbase.alt", ignoring the ALT-to-ref alignment, and labels a potential hit as ALT or non-ALT, depending on whether the hit lands on an ALT contig or not. BWA-MEM then reports alignments and assigns mapQ following these two rules:

• The original mapQ of a non-ALT hit is computed across non-ALT hits only. The reported mapQ of an ALT hit is computed across all hits.

• An ALT hit is only reported if its score is strictly better than all overlapping non-ALT hits. A reported ALT hit is flagged with 0x800 (supplementary) unless there are no non-ALT hits.

When option -g FLOAT is in use (which is the default), a third rule kicks in:

• The mapQ of a non-ALT hit is reduced to zero if its score is less than FLOAT times the score of an overlapping ALT hit. In this case, the original mapQ is moved to the om tag.

If we don't care about ALT hits, we may actually skip postprocessing (step 2). Nonetheless, postprocessing is recommended as it improves mapQ and gives more information about ALT hits.

Step 2: Postprocessing

Postprocessing is done with a separate script bwa-postalt.js. It reads all potential hits reported in the XA tag, lifts ALT hits to the chromosomal positions using the ALT-to-ref alignment, groups them after lifting and then reassigns mapQ based on the best scoring hit in each group with all the hits in a group get the same mapQ. Knowing the ALT-to-ref alignment, this script can greatly improve mapQ of ALT hits and occasionally improve mapQ of non-ALT hits.

The script also measures the presence of each ALT contig. For a group of overlapping ALT contigs c_1, ..., c_m, the weight for c_k equals \frac{\sum_j P(c_k|r_j)}{\sum_j\max_i P(c_i|r_j)}, where P(c_k|r)=\frac{pow(4,s_k)}{\sum_i pow(4,s_i)} is the posterior of c_k given a read r mapped to it with a Smith-Waterman score s_k. This weight is reported in postinfo.ctw in the option 2 above.

On the Completeness of GRCh38+ALT

While GRCh38 is much more complete than GRCh37, it is still missing some true human sequences. To make sure every piece of sequence in the reference assembly is correct, the Genome Reference Consortium (GRC) require each ALT contig to have enough support from multiple sources before considering to add it to the reference assembly. This careful procedure has left out some sequences, one of which is this example, a 10kb contig assembled from CHM1 short reads and present also in NA12878. You can try BLAT or BLAST to see where it maps.

For a more complete reference genome, we compiled a new set of decoy sequences from GenBank clones and the de novo assembly of 254 public SGDP samples. The sequences are included in hs38d4-extra.fa from the BWA resource bundle for GRCh38.

In addition to decoy, we also put multiple alleles of HLA genes in hs38d4-extra.fa. These genomic sequences were acquired from IMGT/HLA, version 3.18.0. Script bwa-postalt.js also helps to genotype HLA genes, though not to high resolution for now.

Problems and Future Development

There are some uncertainties about ALT mappings - we are not sure whether they help biological discovery and don't know the best way to analyze them. Without clear demand from downstream analyses, it is very difficult to design the optimal mapping strategy. The current BWA-MEM method is just a start. If it turns out to be useful in research, we will probably rewrite bwa-postalt.js in C for performance; if not, we will try new designs. It is also possible that we may make breakthrough on the representation of multiple genomes, in which case, we can even get rid of ALT contigs once for all.