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Instructions for the use of transcriptional group comparison software HISAT2

At present, the common analysis process of transcriptome analysis has changed from the combination of Hophat + cufflinks to the combination of HISTA and StringTie. The Protocol of this combination can be found in the article "Transcript-level expression analysis of RNA-seq experiments with HISAT, StringTie and Ballgown" published on Nature Protocol.

First of all, let's take a look at the comparison software HISTA, its speed and accuracy are greatly improved compared with Tophat.

The instructions for use are as follows:

Hisat2 [options] *-x {- 1-2 |-U |-- sra-acc} [- S]

Prefix of the Index file (* .X.ht2)

Read1 file (supports gz,bzip2 compression format)

Read2 file (supports gz,bzip2 compression format)

Output unpaired alignment sequence (supports gz,bzip2 compression format)

Support downloading of NCBI SRA data, using commas to separate different SRA numbers

Compare the output of the resulting SAM file (default: standard output)

You can enter a list of files separated by commas, and multiple entries such as'- U file1.fq,file2.fq-U file3.fq'.

Option (default value in parentheses):

Enter:

-Q input file format is FASTQ .fq / .fastq (default)

-- qseq Q input file format is Illumina's qseq format

-f input file format is FASTA .fa / .mfa with multiple sequences

The-r input is an one-line sequence

-c, are sequences themselves, not files

-s/--skip skips the reads/pairs (none) in front of the input file

-stop the program when u/--upto exceeds the reads/pairs in front of the input file (no limit)

-5/--trim5 removes Reads 5 bases / left base (0)

-3/--trim3 removes the right base (0) of Reads 3cm Universe r

-- phred33 sequence quality value coding is Phred+33 (default encoding format)

-- phred64 sequence quality value coding is Phred+64

-- int-quals sequence quality values are numbers separated by spaces

-- sra-acc SRA login number

Comparison:

-- the proportion of non-A/C/G/Ts allowed by n-ceil in comparisons (Lmem0pence0.15)

-- ignore-quals if the sequencing quality value is ignored, the default quality value is 30 (off)

-- nofw is not compared to positive reads (off)

-- norc is not compared to reverse complementary reads (off)

Cut comparison:

Penalty score of normal splicing site of pen-cansplice (0)

Penalty points for abnormal splicing sites in pen-noncansplice (12)

Penalty function of normal splicing site of pen-canintronlen long intron (Grecamay 8Pol 1)

Penalty function of abnormal splicing sites in pen-noncanintronlen long intron (Grecamay 8pl)

-- minimum intron length of min-intronlen (20)

-- maximum intron length of max-intronlen (500000)

-- known-splicesite-infile specifies a known cut point file

Novel-splicesite-outfile discovers (reports) new splicing sites

-- novel-splicesite-infile specifies some new variable cut sites

-- no-temp-splicesite disable the use of splice sites found

-- no-spliced-alignment disables shearing comparison

-- rna-strandness can only be linked to RNA (unstranded).

-- tmo only reports reads on comparisons with known transcripts

-- dta reports comparison reads specially assembled for transcripts

-- dta-cufflinks reports comparison reads assembled specifically for cufflinks

Score:

-- ma matching score (0 for-- end-to-end, 2 for-- local)

-- mp, maximum and minimum penalty points for locus mismatching, low quality, low penalty points

-- sp, max and min penalties for soft-clipping; lower qual = lower penalty

Penalty points for np non-A/C/G/Ts matches (1)

-- rdg, read penalty points for space opening and extension (5pc3)

-- rfg, penalty points for open and extended spaces in the reference sequence (5pc3)

-- minimum acceptable comparison score for score-min (LMIT 0. 0 MALEMAY 0. 2)

Compare the report output:

(default) compare more results and report only the best ones

OR

-k comparison results, the maximum number of comparisons that can be reported

OR

-the a/--all report compares all the results

Double-end comparison:

-- the direction of fr/--rf/--ff reads alignment fw/rev, rev/fw, fw/fw (--fr)

-- no-mixed does not do unmatched reads comparisons

-- no-discordant compare reads comparison with inconsistent distance

Output:

-t/--time output the time spent during the search

-- reads output path not matched by un

-- reads output path on al one-end comparison

-- reads output path with inconsistent un-conc alignment position

-- al-conc has at least one location alignment consistent reads output path

-- un-gz, to gzip compress output, or add'- bz2' to bzip2 compress output.)

Quiet does not print error output unless there is a serious error

-- met-file saves metrics to file (off)

-- met-stderr print metrics large standard error output (off)

How many seconds does met report internal counters and metrics (1)

-- no-head does not output head information in SAM files

-- no-sq does not output @ SQ information of head in the SAM file

-- rg-id sets reads ID information

-- rg adds reads packet information

Omit-sec-seq put'*'in SEQ and QUAL fields for secondary alignments.

Performance:

-o/--offrate overrides index's offrate

-number of threads compared by p/--threads (1)

-- reorder forces the order of the reads in the output SAM file to be the same as the imported reads

-- mm shares index through memory so that multiple bowtie can be shared

Other:

-- qc-filter filter reads with low quality

-- seed (seed) of random numbers generated by seed (0)

-- non-deterministic random number generation uses seed instead of reads attribute

-- remove-chrname deletes' chr' from the reference sequence name in the alignment result

-- add-chrname adds' chr' to the reference sequence name in the alignment result

-- version outputs version information of the software

-documentation for the use of h/--help output software

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