#
Map Reads onto a genome with BWA MEM
- at least 4 cores/threads available
- a genome assembly in FASTA format: .fasta .fa .fasta.gz .fa.gz
- paired-end fastq sequence files
gzipped recommended
- sample name: a-z 0-9 . _ - case insensitive
- forward: _F .F .1 _1 _R1_001 .R1_001 _R1 .R1
- reverse: _R .R .2 _2 _R2_001 .R2_001 _R2 .R2
- fastq extension: .fq .fastq .FQ .FASTQ
Once sequences have been trimmed and passed through other QC filters, they will need to be aligned to a reference genome. This module within Harpy expects filtered reads as input, such as those derived using harpy qc . You can map reads onto a genome assembly with Harpy using the align bwa module:
harpy align bwa OPTIONS... INPUTS...harpy align bwa --genome genome.fasta Sequences/
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Running Options
In addition to the common runtime options , the align bwa module is configured using these command-line arguments:
#
Molecule distance
The --molecule-distance option is used during the BWA alignment workflow
to assign alignments a unique Molecular Identifier MI:i tag based on their
haplotag barcode and the distance threshold you specify. See
haplotag data for more information on
what this value does.
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Quality filtering
The --min-quality argument filters out alignments below a given MQ threshold. The default, 30, keeps alignments
that are at least 99.9% likely correctly mapped. Set this value to 1 if you only want alignments removed with
MQ = 0 (0% likely correct). You may also set it to 0 to keep all alignments for diagnostic purposes.
The plot below shows the relationship between MQ score and the likelihood the alignment is correct and will serve to help you decide
on a value you may want to use. It is common to remove alignments with MQ <30 (<99.9% chance correct) or MQ <40 (<99.99% chance correct).
Every alignment in a BAM file has an associated mapping quality score (MQ) that informs you of the likelihood that the alignment is accurate. This score can range from 0-40, where higher numbers mean the alignment is more likely correct. The math governing the MQ score actually calculates the percent chance the alignment is incorrect:
\%\ chance\ incorrect = 10^\frac{-MQ}{10} \times 100\\
\text{where }0\le MQ\le 40You can simply subtract it from 100 to determine the percent chance the alignment is correct:
\%\ chance\ correct = 100 - \%\ chance\ incorrect\\
\text{or} \\
\%\ chance\ correct = (1 - 10^\frac{-MQ}{10}) \times 100
#
Marking PCR duplicates
Harpy uses samtools markdup to mark putative PCR duplicates. By using the --barcode-tag BX
option, it considers the linked-read barcode for more accurate duplicate detection. Duplicate
marking also uses the -S option to mark supplementary (chimeric) alignments as duplicates
if the primary alignment was marked as a duplicate. Duplicates get marked but are not removed.
#
BWA workflow
- default aligner
- ignores (but retains) barcode information
- fast
The BWA MEM workflow is much simpler and faster than the EMA workflow
and maps all reads against the reference genome. Duplicates are marked using samtools markdup.
The BX:Z tags in the read headers are still added to the alignment headers, even though barcodes
are not used to inform mapping. The -m threshold is used for alignment molecule assignment.
graph LR
A([index genome]):::clean --> B([align to genome]):::clean
B-->C([sort alignments]):::clean
C-->D([mark duplicates]):::clean
D-->E([assign molecules]):::clean
E-->F([alignment metrics]):::clean
D-->G([barcode stats]):::clean
G-->F
subgraph aln [Inputs]
Z[FASTQ files]:::clean---genome:::clean
end
aln-->B & A
subgraph markdp [mark duplicates via `samtools`]
direction LR
collate:::clean-->fixmate:::clean
fixmate-->sort:::clean
sort-->markdup:::clean
end
style markdp fill:#f0f0f0,stroke:#e8e8e8,stroke-width:2px
style aln fill:#f0f0f0,stroke:#e8e8e8,stroke-width:2px
classDef clean fill:#f5f6f9,stroke:#b7c9ef,stroke-width:2px
The default output directory is Align/bwa with the folder structure below. Sample1 is a generic sample name for demonstration purposes.
The resulting folder also includes a workflow directory (not shown) with workflow-relevant runtime files and information.
Align/bwa
├── Sample1.bam
├── Sample1.bam.bai
├── logs
│ ├── sample1.bwa.log
│ ├── sample1.markdup.log
│ │── sample1.sort.log
└── reports
├── barcodes.summary.html
├── bwa.stats.html
├── Sample1.html
└── data
├── bxstats
│ └── Sample1.bxstats.gz
└── coverage
└── Sample1.cov.gzBy default, Harpy runs bwa with these parameters (excluding inputs and outputs):
bwa mem -C -R "@RG\tID:samplename\tSM:samplename"Below is a list of all bwa mem command line arguments, excluding those Harpy already uses or those made redundant by Harpy's implementation of BWA.
These are taken directly from the BWA documentation.
-k INT Minimum seed length. Matches shorter than INT will be missed. The alignment speed is usually insensitive to this value unless it significantly deviates 20. [19]
-w INT Band width. Essentially, gaps longer than INT will not be found. Note that the maximum gap length is also affected by the scoring matrix and the hit length, not solely determined by this option. [100]
-d INT Off-diagonal X-dropoff (Z-dropoff). Stop extension when the difference between the best and the current extension score is above |i-j|*A+INT, where i and j are the current positions of the query and reference, respectively, and A is the matching score. Z-dropoff is similar to BLAST’s X-dropoff except that it doesn’t penalize gaps in one of the sequences in the alignment. Z-dropoff not only avoids unnecessary extension, but also reduces poor alignments inside a long good alignment. [100]
-r FLOAT Trigger re-seeding for a MEM longer than minSeedLen*FLOAT. This is a key heuristic parameter for tuning the performance. Larger value yields fewer seeds, which leads to faster alignment speed but lower accuracy. [1.5]
-c INT Discard a MEM if it has more than INT occurence in the genome. This is an insensitive parameter. [10000]
-P In the paired-end mode, perform SW to rescue missing hits only but do not try to find hits that fit a proper pair.
-A INT Matching score. [1]
-B INT Mismatch penalty. The sequence error rate is approximately: {.75 * exp[-log(4) * B/A]}. [4]
-O INT Gap open penalty. [6]
-E INT Gap extension penalty. A gap of length k costs O + k*E (i.e. -O is for opening a zero-length gap). [1]
-L INT Clipping penalty. When performing SW extension, BWA-MEM keeps track of the best score reaching the end of query. If this score is larger than the best SW score minus the clipping penalty, clipping will not be applied. Note that in this case, the SAM AS tag reports the best SW score; clipping penalty is not deducted. [5]
-U INT Penalty for an unpaired read pair. BWA-MEM scores an unpaired read pair as scoreRead1+scoreRead2-INT and scores a paired as scoreRead1+scoreRead2-insertPenalty. It compares these two scores to determine whether we should force pairing. [9]
-T INT Don’t output alignment with score lower than INT. This option only affects output. [30]
-a Output all found alignments for single-end or unpaired paired-end reads. These alignments will be flagged as secondary alignments.
-H Use hard clipping ’H’ in the SAM output. This option may dramatically reduce the redundancy of output when mapping long contig or BAC sequences.These are the summary reports Harpy generates for this workflow. You may right-click the images and open them in a new tab if you wish to see the examples in better detail.
An aggregate report of barcode-specific alignment information for all samples.
Reports the inferred molecule sized based on barcodes in the alignments.
Reports the general statistics computed by samtools stats and flagstat
