root 14:49:03 ~
$ conda activate test
(test) root 14:49:14 ~
$ cd bio
-bash: cd: bio: No such file or directory
(test) root 14:49:18 ~
$ cd biosoft/
(test) root 14:49:23 ~/biosoft
$ ls
bget_0.3.2_Linux_64-bit.tar.gz hisat2-2.2.1-Linux_x86_64.zip
download index.html
(test) root 14:49:46 ~/biosoft
$ unzip hisat2-2.2.1-Linux_x86_64.zip
Archive: hisat2-2.2.1-Linux_x86_64.zip
creating: hisat2-2.2.1/
inflating: hisat2-2.2.1/hisat2-align-s-debug
inflating: hisat2-2.2.1/hisat2_extract_snps_haplotypes_VCF.py
inflating: hisat2-2.2.1/hisat2-build-l
inflating: hisat2-2.2.1/hisat2_extract_exons.py
inflating: hisat2-2.2.1/MANUAL.markdown
creating: hisat2-2.2.1/example/
creating: hisat2-2.2.1/example/index/
inflating: hisat2-2.2.1/example/index/22_20-21M_snp.7.ht2
inflating: hisat2-2.2.1/example/index/22_20-21M_snp.6.ht2
inflating: hisat2-2.2.1/example/index/22_20-21M_snp.1.ht2
inflating: hisat2-2.2.1/example/index/22_20-21M_snp.3.ht2
inflating: hisat2-2.2.1/example/index/22_20-21M_snp.5.ht2
inflating: hisat2-2.2.1/example/index/22_20-21M_snp.2.ht2
inflating: hisat2-2.2.1/example/index/22_20-21M_snp.8.ht2
inflating: hisat2-2.2.1/example/index/22_20-21M_snp.4.ht2
creating: hisat2-2.2.1/example/reference/
inflating: hisat2-2.2.1/example/reference/22_20-21M.snp
inflating: hisat2-2.2.1/example/reference/22_20-21M.fa
creating: hisat2-2.2.1/example/reads/
inflating: hisat2-2.2.1/example/reads/reads_1.fa
inflating: hisat2-2.2.1/example/reads/reads_2.fa
inflating: hisat2-2.2.1/hisat2-build-l-debug
inflating: hisat2-2.2.1/hisat2-inspect-l
inflating: hisat2-2.2.1/LICENSE
inflating: hisat2-2.2.1/hisat2-align-l
inflating: hisat2-2.2.1/hisat2-inspect-l-debug
inflating: hisat2-2.2.1/hisat2-inspect-s-debug
inflating: hisat2-2.2.1/hisat2
inflating: hisat2-2.2.1/extract_splice_sites.py
inflating: hisat2-2.2.1/hisat2-align-s
inflating: hisat2-2.2.1/hisat2-inspect
inflating: hisat2-2.2.1/hisat2_simulate_reads.py
inflating: hisat2-2.2.1/AUTHORS
creating: hisat2-2.2.1/scripts/
inflating: hisat2-2.2.1/scripts/make_sc3.sh
inflating: hisat2-2.2.1/scripts/make_grch37_tran.sh
inflating: hisat2-2.2.1/scripts/make_e_coli.sh
inflating: hisat2-2.2.1/scripts/make_rnor6_tran.sh
inflating: hisat2-2.2.1/scripts/make_hg19.sh
inflating: hisat2-2.2.1/scripts/make_mm10.sh
inflating: hisat2-2.2.1/scripts/make_grch38_snp.sh
inflating: hisat2-2.2.1/scripts/make_rnor6.sh
inflating: hisat2-2.2.1/scripts/make_grcm38_tran.sh
inflating: hisat2-2.2.1/scripts/make_zm3_snp_tran_ercc.sh
inflating: hisat2-2.2.1/scripts/make_bdgp6.sh
inflating: hisat2-2.2.1/scripts/make_dm6.sh
inflating: hisat2-2.2.1/scripts/make_r64.sh
inflating: hisat2-2.2.1/scripts/make_bdgp6_tran.sh
inflating: hisat2-2.2.1/scripts/make_grch38_snp_rep.sh
inflating: hisat2-2.2.1/scripts/infer_fraglen.pl
inflating: hisat2-2.2.1/scripts/make_grch38_rep.sh
inflating: hisat2-2.2.1/scripts/make_grch38_snp_tran.sh
inflating: hisat2-2.2.1/scripts/make_grch37.sh
inflating: hisat2-2.2.1/scripts/make_wbcel235_tran.sh
inflating: hisat2-2.2.1/scripts/make_grcm38_snp.sh
inflating: hisat2-2.2.1/scripts/make_grch38.sh
inflating: hisat2-2.2.1/scripts/make_rn4.sh
inflating: hisat2-2.2.1/scripts/make_hg38_snp.sh
inflating: hisat2-2.2.1/scripts/gen_occ_lookup.pl
inflating: hisat2-2.2.1/scripts/gen_2b_occ_lookup.pl
inflating: hisat2-2.2.1/scripts/make_rn6.sh
inflating: hisat2-2.2.1/scripts/make_hg38.sh
inflating: hisat2-2.2.1/scripts/make_ce10.sh
inflating: hisat2-2.2.1/scripts/make_wbcel235.sh
inflating: hisat2-2.2.1/scripts/make_grcm38_snp_tran.sh
inflating: hisat2-2.2.1/scripts/make_grch37_snp_tran.sh
inflating: hisat2-2.2.1/scripts/make_mm9.sh
inflating: hisat2-2.2.1/scripts/make_hg38_snp_tran.sh
inflating: hisat2-2.2.1/scripts/make_grch37_snp_tran_ercc.sh
inflating: hisat2-2.2.1/scripts/convert_quals.pl
inflating: hisat2-2.2.1/scripts/make_r64_tran.sh
inflating: hisat2-2.2.1/scripts/make_canFam2.sh
inflating: hisat2-2.2.1/scripts/make_grch38_snp_tran_ercc.sh
inflating: hisat2-2.2.1/scripts/gen_solqual_lookup.pl
inflating: hisat2-2.2.1/scripts/make_hg38_allsnp.sh
inflating: hisat2-2.2.1/scripts/make_a_thaliana_tair.sh
inflating: hisat2-2.2.1/scripts/make_grch38_tran.sh
inflating: hisat2-2.2.1/scripts/make_b_taurus_UMD3.sh
inflating: hisat2-2.2.1/scripts/make_grcm38.sh
inflating: hisat2-2.2.1/scripts/make_grch37_snp.sh
inflating: hisat2-2.2.1/hisat2-build-s
inflating: hisat2-2.2.1/hisat2-repeat
inflating: hisat2-2.2.1/TUTORIAL
inflating: hisat2-2.2.1/hisat2-inspect-s
inflating: hisat2-2.2.1/hisat2_read_statistics.py
inflating: hisat2-2.2.1/hisat2-build-s-debug
inflating: hisat2-2.2.1/hisat2-align-l-debug
inflating: hisat2-2.2.1/extract_exons.py
inflating: hisat2-2.2.1/hisat2_extract_snps_haplotypes_UCSC.py
inflating: hisat2-2.2.1/NEWS
inflating: hisat2-2.2.1/hisat2_extract_splice_sites.py
extracting: hisat2-2.2.1/VERSION
inflating: hisat2-2.2.1/MANUAL
inflating: hisat2-2.2.1/hisat2-repeat-debug
inflating: hisat2-2.2.1/hisat2-build
(test) root 14:50:11 ~/biosoft
$ ls
bget_0.3.2_Linux_64-bit.tar.gz hisat2-2.2.1 index.html
download hisat2-2.2.1-Linux_x86_64.zip
(test) root 14:50:47 ~/biosoft
$ cd hisat2-2.2.1
(test) root 14:51:25 ~/biosoft/hisat2-2.2.1
$ ls
AUTHORS hisat2_extract_splice_sites.py
example hisat2-inspect
extract_exons.py hisat2-inspect-l
extract_splice_sites.py hisat2-inspect-l-debug
hisat2 hisat2-inspect-s
hisat2-align-l hisat2-inspect-s-debug
hisat2-align-l-debug hisat2_read_statistics.py
hisat2-align-s hisat2-repeat
hisat2-align-s-debug hisat2-repeat-debug
hisat2-build hisat2_simulate_reads.py
hisat2-build-l LICENSE
hisat2-build-l-debug MANUAL
hisat2-build-s MANUAL.markdown
hisat2-build-s-debug NEWS
hisat2_extract_exons.py scripts
hisat2_extract_snps_haplotypes_UCSC.py TUTORIAL
hisat2_extract_snps_haplotypes_VCF.py VERSION
(test) root 14:51:33 ~/biosoft/hisat2-2.2.1
$ hisat2
-bash: hisat2: command not found
(test) root 14:51:40 ~/biosoft/hisat2-2.2.1
$ hisat2 --help
-bash: hisat2: command not found
(test) root 14:52:03 ~/biosoft/hisat2-2.2.1
$ ./hisat2 --help
HISAT2 version 2.2.1 by Daehwan Kim ([email protected], www.ccb.jhu.edu/people/infphilo)
Usage:
hisat2 [options]* -x {-1 -2 | -U | --sra-acc } [-S ]
Index filename prefix (minus trailing .X.ht2).
Files with #1 mates, paired with files in .
Could be gzip'ed (extension: .gz) or bzip2'ed (extension: .bz2).
Files with #2 mates, paired with files in .
Could be gzip'ed (extension: .gz) or bzip2'ed (extension: .bz2).
Files with unpaired reads.
Could be gzip'ed (extension: .gz) or bzip2'ed (extension: .bz2).
Comma-separated list of SRA accession numbers, e.g. --sra-acc SRR353653,SRR353654.
File for SAM output (default: stdout)
, , 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 , , are sequences themselves, not files
-s/--skip skip the first reads/pairs in the input (none)
-u/--upto stop after first reads/pairs (no limit)
-5/--trim5 trim bases from 5'/left end of reads (0)
-3/--trim3 trim 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
--sra-acc SRA accession ID
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 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 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 penalty for a canonical splice site (0)
--pen-noncansplice penalty for a non-canonical splice site (12)
--pen-canintronlen penalty for long introns (G,-8,1) with canonical splice sites
--pen-noncanintronlen penalty for long introns (G,-8,1) with noncanonical splice sites
--min-intronlen minimum intron length (20)
--max-intronlen maximum intron length (500000)
--known-splicesite-infile provide a list of known splice sites
--novel-splicesite-outfile report a list of splice sites
--novel-splicesite-infile 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 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 , max and min penalties for mismatch; lower qual = lower penalty <6,2>
--sp , max and min penalties for soft-clipping; lower qual = lower penalty <2,1>
--no-softclip no soft-clipping
--np penalty for non-A/C/G/Ts in read/ref (1)
--rdg , read gap open, extend penalties (5,3)
--rfg , reference gap open, extend penalties (5,3)
--score-min min acceptable alignment score w/r/t read length
(L,0.0,-0.2)
Reporting:
-k It searches for at most 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 , 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 distinct,
valid alignments, hisat2 does not guarantee that the 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 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 minimum fragment length (0), only valid with --no-spliced-alignment
-X/--maxins 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 write unpaired reads that didn't align to
--al write unpaired reads that aligned at least once to
--un-conc write pairs that didn't align concordantly to
--al-conc write pairs that aligned concordantly at least once to
(Note: for --un, --al, --un-conc, or --al-conc, add '-gz' to the option name, e.g.
--un-gz , to gzip compress output, or add '-bz2' to bzip2 compress output.)
--summary-file 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 send metrics to file at (off)
--met-stderr send metrics to stderr (off)
--met report internal counters & metrics every secs (1)
--no-head suppress header lines, i.e. lines starting with @
--no-sq suppress @SQ header lines
--rg-id set read group id, reflected in @RG line and RG:Z: opt field
--rg add ("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 override offrate of index; must be >= index's offrate
-p/--threads 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 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
(test) root 14:52:13 ~/biosoft/hisat2-2.2.1
(test) root 14:52:13 ~/biosoft/hisat2-2.2.1
$ PATH=root/biosoft/hisat2-2.2.1/:$PATH
(test) root 14:55:42 ~/biosoft/hisat2-2.2.1
$ hisat2 --help
-bash: hisat2: command not found
(test) root 14:56:00 ~/biosoft/hisat2-2.2.1
$ PATH=root/biosoft/hisat2-2.2.1/:$PATH
(test) root 14:56:31 ~/biosoft/hisat2-2.2.1
$ PATH=/root/biosoft/hisat2-2.2.1/:$PATH
(test) root 14:56:57 ~/biosoft/hisat2-2.2.1
$ hisat2 --help
HISAT2 version 2.2.1 by Daehwan Kim ([email protected], www.ccb.jhu.edu/people/infphilo)
Usage:
hisat2 [options]* -x {-1 -2 | -U | --sra-acc } [-S ]
Index filename prefix (minus trailing .X.ht2).
Files with #1 mates, paired with files in .
Could be gzip'ed (extension: .gz) or bzip2'ed (extension: .bz2).
Files with #2 mates, paired with files in .
Could be gzip'ed (extension: .gz) or bzip2'ed (extension: .bz2).
Files with unpaired reads.
Could be gzip'ed (extension: .gz) or bzip2'ed (extension: .bz2).
Comma-separated list of SRA accession numbers, e.g. --sra-acc SRR353653,SRR353654.
File for SAM output (default: stdout)
, , 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 , , are sequences themselves, not files
-s/--skip skip the first reads/pairs in the input (none)
-u/--upto stop after first reads/pairs (no limit)
-5/--trim5 trim bases from 5'/left end of reads (0)
-3/--trim3 trim 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
--sra-acc SRA accession ID
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 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 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 penalty for a canonical splice site (0)
--pen-noncansplice penalty for a non-canonical splice site (12)
--pen-canintronlen penalty for long introns (G,-8,1) with canonical splice sites
--pen-noncanintronlen penalty for long introns (G,-8,1) with noncanonical splice sites
--min-intronlen minimum intron length (20)
--max-intronlen maximum intron length (500000)
--known-splicesite-infile provide a list of known splice sites
--novel-splicesite-outfile report a list of splice sites
--novel-splicesite-infile 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 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 , max and min penalties for mismatch; lower qual = lower penalty <6,2>
--sp , max and min penalties for soft-clipping; lower qual = lower penalty <2,1>
--no-softclip no soft-clipping
--np penalty for non-A/C/G/Ts in read/ref (1)
--rdg , read gap open, extend penalties (5,3)
--rfg , reference gap open, extend penalties (5,3)
--score-min min acceptable alignment score w/r/t read length
(L,0.0,-0.2)
Reporting:
-k It searches for at most 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 , 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 distinct,
valid alignments, hisat2 does not guarantee that the 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 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 minimum fragment length (0), only valid with --no-spliced-alignment
-X/--maxins 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 write unpaired reads that didn't align to
--al write unpaired reads that aligned at least once to
--un-conc write pairs that didn't align concordantly to
--al-conc write pairs that aligned concordantly at least once to
(Note: for --un, --al, --un-conc, or --al-conc, add '-gz' to the option name, e.g.
--un-gz , to gzip compress output, or add '-bz2' to bzip2 compress output.)
--summary-file 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 send metrics to file at (off)
--met-stderr send metrics to stderr (off)
--met report internal counters & metrics every secs (1)
--no-head suppress header lines, i.e. lines starting with @
--no-sq suppress @SQ header lines
--rg-id set read group id, reflected in @RG line and RG:Z: opt field
--rg add ("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 override offrate of index; must be >= index's offrate
-p/--threads 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 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
(test) root 14:57:04 ~/biosoft/hisat2-2.2.1
