Single-end alignment

Single-end (SE) alignment maps each read in a single FASTQ file independently — there’s no mate pair to constrain the alignment.

Basic command

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

Strand information

For stranded RNA-seq libraries, add the strand field to the output so that downstream tools (Cufflinks, StringTie, etc.) can pick up the strand assignment from junction motifs:

--outSAMstrandField intronMotif

Gzipped input

Use --readFilesCommand zcat to pipe through decompression:

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

On macOS, use gzcat instead of zcat.

Threading

Use --runThreadN to parallelise the alignment:

--runThreadN 16

The alignment phase scales nearly linearly with thread count up to the IO limit of the storage holding the input/output files.

Filtering

The defaults match STAR. Two parameters are particularly useful to know:

• --outFilterMultimapNmax (default 10) — max number of alignments per read. A read mapping to more loci is discarded as MultiMapTooMany.
• --outFilterMismatchNoverLmax (default 0.3) — max ratio of mismatches to mapped length. Tightening this to 0.04 is a common choice for human RNA-seq.
• --outFilterScoreMinOverLread (default 0.66) — min alignment score relative to read length. Reads scoring below this threshold are unmapped.

See the CLI parameters reference for the full list.

Output limits

Multi-mapping reads emit one SAM record per locus by default (up to --outFilterMultimapNmax). To cap the number of secondary alignments written:

--outSAMmultNmax 5

Reproducibility

When two alignments tie on score, rustar-aligner uses a seeded RNG (StdRng) to pick the primary. Set the seed for reproducible runs:

--runRNGseed 42

The default seed is 777.

What’s next

• Paired-end alignment for paired libraries
• Two-pass mode for novel-junction recovery
• Output files reference for what each output file contains