NAME¶

hisat2-align-s - graph-based alignment of short nucleotide reads to many genomes, wrapper script

DESCRIPTION¶

HISAT2 version 2.2.1 by Daehwan Kim (infphilo@gmail.com, www.ccb.jhu.edu/people/infphilo) Usage:

hisat2 [options]* -x <ht2-idx> {-1 <m1> -2 <m2> | -U <r>} [-S <sam>]

<ht2-idx>

Index filename prefix (minus trailing .X.ht2).

<m1>

Files with #1 mates, paired with files in <m2>. Could be gzip'ed (extension: .gz) or bzip2'ed (extension: .bz2).

<m2>

Files with #2 mates, paired with files in <m1>. Could be gzip'ed (extension: .gz) or bzip2'ed (extension: .bz2).

<r>

Files with unpaired reads. Could be gzip'ed (extension: .gz) or bzip2'ed (extension: .bz2).

<sam>

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):

Input:

-q

query input files are FASTQ .fq/.fastq (default)

--qseq

query input files are in Illumina's qseq format

-f

query input files are (multi-)FASTA .fa/.mfa

-r

query input files are raw one-sequence-per-line

-c

<m1>, <m2>, <r> are sequences themselves, not files

-s/--skip <int>

skip the first <int> reads/pairs in the input (none)

-u/--upto <int>

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)

--phred33

qualities are Phred+33 (default)

--phred64

qualities are Phred+64

--int-quals

qualities encoded as space-delimited integers

Presets:

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

Alignment:

--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)

--n-ceil <func>

func for max # non-A/C/G/Ts permitted in aln (L,0,0.15)

--ignore-quals

treat all quality values as 30 on Phred scale (off)

--nofw

do not align forward (original) version of read (off)

--norc

do not align reverse-complement version of read (off)

--no-repeat-index

do not use repeat index

Spliced Alignment:

--pen-cansplice <int>

penalty for a canonical splice site (0)

--pen-noncansplice <int>

penalty for a non-canonical splice site (12)

--pen-canintronlen <func>

penalty for long introns (G,-8,1) with canonical splice sites

--pen-noncanintronlen <func>

penalty for long introns (G,-8,1) with noncanonical splice sites

--min-intronlen <int>

minimum intron length (20)

--max-intronlen <int>

maximum intron length (500000)

--known-splicesite-infile <path>

provide a list of known splice sites

--novel-splicesite-outfile <path>

report a list of splice sites

--novel-splicesite-infile <path>

provide a list of novel splice sites

--no-temp-splicesite

disable the use of splice sites found

--no-spliced-alignment

disable spliced alignment

--rna-strandness <string>

specify strand-specific information (unstranded)

--tmo

reports only those alignments within known transcriptome

--dta

reports alignments tailored for transcript assemblers

--dta-cufflinks

reports alignments tailored specifically for cufflinks

--avoid-pseudogene

tries to avoid aligning reads to pseudogenes (experimental option)

--no-templatelen-adjustment

disables template length adjustment for RNA-seq reads

Scoring:

--mp <int>,<int>

max and min penalties for mismatch; lower qual = lower penalty <6,2>

--sp <int>,<int>

max and min penalties for soft-clipping; lower qual = lower penalty <2,1>

--no-softclip

no soft-clipping

--np <int>

penalty for non-A/C/G/Ts in read/ref (1)

--rdg <int>,<int>

read gap open, extend penalties (5,3)

--rfg <int>,<int>

reference gap open, extend penalties (5,3)

--score-min <func> min acceptable alignment score w/r/t read length

(L,0.0,-0.2)

Reporting:

-k <int>

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.

--max-seeds <int>

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.

-a/--all

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).

--repeat

report alignments to repeat sequences directly

Paired-end:

-I/--minins <int>

minimum fragment length (0), only valid with --no-spliced-alignment

-X/--maxins <int>

maximum fragment length (500), only valid with --no-spliced-alignment

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

--no-mixed

suppress unpaired alignments for paired reads

--no-discordant

suppress discordant alignments for paired reads

Output:

-t/--time

print wall-clock time taken by search phases

--un <path>

write unpaired reads that didn't align to <path>

--al <path>

write unpaired reads that aligned at least once to <path>

--un-conc <path>

write pairs that didn't align concordantly to <path>

--al-conc <path>

write pairs that aligned concordantly at least once to <path>

(Note: for --un, --al, --un-conc, or --al-conc, add '-gz' to the option name, e.g. --un-gz <path>, to gzip compress output, or add '-bz2' to bzip2 compress output.) --summary-file <path> print alignment summary to this file. --new-summary print alignment summary in a new style, which is more machine-friendly. --quiet print nothing to stderr except serious errors --met-file <path> send metrics to file at <path> (off) --met-stderr send metrics to stderr (off) --met <int> report internal counters & metrics every <int> secs (1) --no-head suppress header lines, i.e. lines starting with @ --no-sq suppress @SQ header lines --rg-id <text> set read group id, reflected in @RG line and RG:Z: opt field --rg <text> add <text> ("lab:value") to @RG line of SAM header.

Note: @RG line only printed when --rg-id is set.

--omit-sec-seq

put '*' in SEQ and QUAL fields for secondary alignments.

Performance:

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

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

--reorder

force SAM output order to match order of input reads

--mm

use memory-mapped I/O for index; many 'hisat2's can share

Other:

--qc-filter

filter out reads that are bad according to QSEQ filter

--seed <int>

seed for random number generator (0)

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

--remove-chrname

remove 'chr' from reference names in alignment

--add-chrname

add 'chr' to reference names in alignment

--version

print version information and quit

-h/--help

print this usage message

64-bit Built on Debian 25 February 2023 Compiler: gcc version 12.2.0 (Debian 12.2.0-14) 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}