Paired-end alignment

Paired-end (PE) alignment maps both mates of a fragment together, using the expected insert size and pair geometry to constrain the alignment. rustar-aligner handles mate rescue (recovering one mate when only the other anchors), proper-pair flagging, and reports NH / HI tags consistent with STAR’s PE pipeline.

Basic command

Pass both FASTQ files to --readFilesIn. Mate 1 first, mate 2 second — order matters.

rustar-aligner \
  --genomeDir /path/to/genome_index \
  --readFilesIn reads_1.fq reads_2.fq \
  --outSAMtype SAM \
  --outFileNamePrefix sample_

Gzipped input

rustar-aligner \
  --genomeDir /path/to/genome_index \
  --readFilesIn reads_1.fq.gz reads_2.fq.gz \
  --readFilesCommand zcat \
  --outSAMtype BAM SortedByCoordinate \
  --outFileNamePrefix sample_

Insert size

By default rustar-aligner computes the maximum mate gap from the genome size and winBinNbits / winAnchorDistNbins. To set an explicit cap:

--alignMatesGapMax 1000000

This is the maximum genomic distance between the leftmost ends of the two mates. The default is suitable for most RNA-seq libraries.

Spliced mate constraints

Two parameters control the minimum mapped length of a spliced mate. They were both introduced because, for poorly-anchored mates, STAR (and rustar-aligner) can otherwise emit very short, low-confidence spliced alignments:

• --alignSplicedMateMapLmin (absolute, default 0 = off) — minimum number of bases that must be mapped on a spliced mate.
• --alignSplicedMateMapLminOverLmate (default 0.66) — same, but expressed as a fraction of read length.

The default config means a spliced mate must map at least 66% of its bases, with no absolute floor.

Unmapped mates

By default unmapped mates aren’t written into the SAM/BAM output. To include them in the alignment file:

--outSAMunmapped Within

Or to also keep pairs together when only one mate is mapped:

--outSAMunmapped Within KeepPairs

Or to write unmapped reads to separate FASTQ files:

--outReadsUnmapped Fastx

This produces <prefix>Unmapped.out.mate1 and <prefix>Unmapped.out.mate2.

Half-mapped pairs

If only one mate has a confident alignment, rustar-aligner still emits a record for the other mate (flagged unmapped) so the pair stays together in the SAM/BAM. On the yeast benchmark (10,000 paired reads) rustar-aligner produces 8,390 fully-mapped pairs and zero half-mapped pairs, matching STAR exactly.

Read groups

If you want @RG headers on the output (e.g. for downstream tools that group by sample), supply them with --outSAMattrRGline:

--outSAMattrRGline ID:sample1 SM:sample1 LB:lib1 PL:ILLUMINA

For multi-lane runs, separate RG blocks with a literal ,:

--outSAMattrRGline ID:lane1 SM:sample1 , ID:lane2 SM:sample1

What’s next

• Two-pass mode for novel-junction recovery
• Chimeric detection for fusion calling on PE data
• STAR compatibility report for the PE faithfulness numbers