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HISAT2-ALIGN-L(1) User Commands HISAT2-ALIGN-L(1)


hisat2-align-l - HISAT2 graph-based alignment of short nucleotide reads to many genomes, large index binary


HISAT2 version 2.2.1 by Daehwan Kim (, Usage:

hisat2-align [options]* -x <ht2-idx> {-1 <m1> -2 <m2> | -U <r>} [-S <sam>]
Index filename prefix (minus trailing .X.ht2l).
Files with #1 mates, paired with files in <m2>.
Files with #2 mates, paired with files in <m1>.
Files with unpaired reads.
File for SAM output (default: stdout)
<m1>, <m2>, <r> can be comma-separated lists (no whitespace) and can be specified many times. E.g. '-U file1.fq,file2.fq -U file3.fq'.

Options (defaults in parentheses):

query input files are FASTQ .fq/.fastq (default)
query input files are in Illumina's qseq format
query input files are (multi-)FASTA .fa/.mfa
query input files are raw one-sequence-per-line
<m1>, <m2>, <r> are sequences themselves, not files
skip the first <int> reads/pairs in the input (none)
stop after first <int> reads/pairs (no limit)
-5/--trim5 <int>
trim <int> bases from 5'/left end of reads (0)
-3/--trim3 <int>
trim <int> bases from 3'/right end of reads (0)
qualities are Phred+33 (default)
qualities are Phred+64
qualities encoded as space-delimited integers
Same as:

--fast --no-repeat-index

--sensitive --bowtie2-dp 1 -k 30 --score-min L,0,-0.5

--very-sensitive --bowtie2-dp 2 -k 50 --score-min L,0,-1


--bowtie2-dp <int> use Bowtie2's dynamic programming alignment algorithm (0) - 0: no dynamic programming, 1: conditional dynamic programming, and 2: unconditional dynamic programming (slowest)

func for max # non-A/C/G/Ts permitted in aln (L,0,0.15)
treat all quality values as 30 on Phred scale (off)
do not align forward (original) version of read (off)
do not align reverse-complement version of read (off)
do not use repeat index
Spliced Alignment:
penalty for a canonical splice site (0)
penalty for a non-canonical splice site (12)
penalty for long introns (G,-8,1) with canonical splice sites
penalty for long introns (G,-8,1) with noncanonical splice sites
minimum intron length (20)
maximum intron length (500000)
provide a list of known splice sites
report a list of splice sites
provide a list of novel splice sites
disable the use of splice sites found
disable spliced alignment
specify strand-specific information (unstranded)
reports only those alignments within known transcriptome
reports alignments tailored for transcript assemblers
reports alignments tailored specifically for cufflinks
tries to avoid aligning reads to pseudogenes (experimental option)
disables template length adjustment for RNA-seq reads
max and min penalties for mismatch; lower qual = lower penalty <6,2>
max and min penalties for soft-clipping; lower qual = lower penalty <2,1>
no soft-clipping
penalty for non-A/C/G/Ts in read/ref (1)
read gap open, extend penalties (5,3)
reference gap open, extend penalties (5,3)
It searches for at most <int> distinct, primary alignments for each read. Primary alignments mean alignments whose alignment score is equal to or higher than any other alignments. The search terminates when it cannot find more distinct valid alignments, or when it finds <int>, whichever happens first. The alignment score for a paired-end alignment equals the sum of the alignment scores of the individual mates. Each reported read or pair alignment beyond the first has the SAM ???secondary??? bit (which equals 256) set in its FLAGS field. For reads that have more than <int> distinct, valid alignments, hisat2 does not guarantee that the <int> alignments reported are the best possible in terms of alignment score. Default: 5 (linear index) or 10 (graph index). Note: HISAT2 is not designed with large values for -k in mind, and when aligning reads to long, repetitive genomes, large -k could make alignment much slower.
HISAT2, like other aligners, uses seed-and-extend approaches. HISAT2 tries to extend seeds to full-length alignments. In HISAT2, --max-seeds is used to control the maximum number of seeds that will be extended. For DNA-read alignment (--no-spliced-alignment), HISAT2 extends up to these many seeds and skips the rest of the seeds. For RNA-read alignment, HISAT2 skips extending seeds and reports no alignments if the number of seeds is larger than the number specified with the option, to be compatible with previous versions of HISAT2. Large values for --max-seeds may improve alignment sensitivity, but HISAT2 is not designed with large values for --max-seeds in mind, and when aligning reads to long, repetitive genomes, large --max-seeds could make alignment much slower. The default value is the maximum of 5 and the value that comes with -k times 2.
HISAT2 reports all alignments it can find. Using the option is equivalent to using both --max-seeds and -k with the maximum value that a 64-bit signed integer can represent (9,223,372,036,854,775,807).
report alignments to repeat sequences directly
minimum fragment length (0), only valid with --no-spliced-alignment
maximum fragment length (500), only valid with --no-spliced-alignment

--fr/--rf/--ff -1, -2 mates align fw/rev, rev/fw, fw/fw (--fr)

suppress unpaired alignments for paired reads
suppress discordant alignments for paired reads
print wall-clock time taken by search phases

--summary-file <path> print alignment summary to this file.

print alignment summary in a new style, which is more machine-friendly.
print nothing to stderr except serious errors
send metrics to file at <path> (off)
send metrics to stderr (off)
report internal counters & metrics every <int> secs (1)
suppress header lines, i.e. lines starting with @
suppress @SQ header lines
set read group id, reflected in @RG line and RG:Z: opt field
add <text> ("lab:value") to @RG line of SAM header. Note: @RG line only printed when --rg-id is set.
put '*' in SEQ and QUAL fields for secondary alignments.

-o/--offrate <int> override offrate of index; must be >= index's offrate

-p/--threads <int> number of alignment threads to launch (1)

force SAM output order to match order of input reads
use memory-mapped I/O for index; many 'hisat2's can share
filter out reads that are bad according to QSEQ filter
seed for random number generator (0)

--non-deterministic seed rand. gen. arbitrarily instead of using read attributes

remove 'chr' from reference names in alignment
add 'chr' to reference names in alignment
print version information and quit
print this usage message

64-bit Built on Debian 05 August 2021 Compiler: gcc version 10.2.1 20210110 (Debian 10.2.1-6) Options: -O3 -funroll-loops -g3 -Wdate-time -D_FORTIFY_SOURCE=2 -std=c++11 Sizeof {int, long, long long, void*, size_t, off_t}: {4, 8, 8, 8, 8, 8}

August 2021 hisat2-align-l version 2.2.1