pipelines-docs

Alignment with pbmm2

The pipeline uses pbmm2 to align each unaligned BAM file to the reference genome. The software also sorts the reads by genomic coordinates, strips unnecessary tags, and links methylation tags if present. An integrity check is then performed on the resulting BAM file.

pbmm2 is used to align both the Full Length Non Chimeric (FLNC) reads and the consensus transcripts generated by cluster2.

Aligning and Sorting

Align and sort reads

pbmm2 align --preset ISOSEQ --sort --strip --unmapped reference.fasta unaligned.bam aligned.bam

Arguments:

--preset: use parameters optimized for Iso-Seq data (ISOSEQ).
--sort: sort the aligned reads by genomic coordinates.
--strip: remove extraneous tags if present in the input BAM file. Tags removed: dq, dt, ip, iq, mq, pa, pc, pd, pe, pg, pm, pq, pt, pv, pw, px, sf, sq, st.
--unmapped: retain unmapped reads.

Integrity Check

To confirm the integrity of the alignment BAM file, in-house Python code checks for the presence of the 28-byte empty block representing the EOF marker in BAM format.

Implementation with pbmm2

The pipeline uses pbmm2 version 1.13.0, which wraps minimap2 version 2.26. It’s important to note that pbmm2 sets some defaults that may differ from the standard minimap2.

Default set by pbmm2 for minimap2:

Soft clipping is enabled with -Y.
Long cigars for the CG tag are set using -L.
X/= cigars are used instead of M with --eqx.
Overlapping query intervals with repeated matches trimming are disabled.
Secondary alignments are excluded with --secondary=no.

Note: Due to multi-threading the output alignment ordering can differ between multiple runs with the same input parameters. The same can occur even with option --sort for records that align to the same target sequence, the same position within that target, and in the same orientation, which are the only fields that samtools sort uses.

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