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STAR-转录组比对

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Symbols count in article: 3.4k Reading time ≈ 3 mins.

STAR

简单记录STAR的常用命令。

Index

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STAR --runMode genomeGenerate \
	--genomeDir /path/to/store/index/ \
	--genomeFastaFiles /path/to/ref \
	--runThreadN <processorsN> \
	--sjdbGTFfile /path/to/gtf

--sjdbGTFfile improve alternative splicing detection

--genomeFastaFile: location of the reference sequence that were used to generate index

--genomeDir: location for storing index, directory is preferable

With limit computional resource

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STAR --runThreadN 32 \
	--runMode genomeGenerate \
	--genomeDir /path/to/store/index/ \
    --genomeFastaFiles /path/to/ref \ 
    --limitGenomeGenerateRAM 96000000000  \
    --genomeChrBinNbits 16

add --limitGenomeGenerateRAM to specify RAM when STAR terminated by small RAM

add --genomeChrBinNbits when STAR encounters error: terminate called after throwing an instance of 'std::bad_alloc'

from https://github.com/alexdobin/STAR/issues/103#issuecomment-173009628

If you are using a genome with a large > 5,000 number of references (chrosomes/scaffolds), you may need to reduce the –{genomeChrBinNbits to reduce RAM consumption. The following scaling is recommended: –genomeChrBinNbits} = min(18, log2(GenomeLength/NumberOfReferences)). For example, for 3~gigaBase genome with 100,000 chromosomes/scaffolds, this is equal to 15.

Aligning

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# paired-end mode
if ${PAIR}; then
	STAR --genomeDir /path/to/index \
	--readFilesIn <fq1> <fq2> \
	--runThreadN <processorsN> \
	--outSAMtype SAM \
	--outFileNamePrefix /path/to/output
else # single-end mode
	STAR --genomeDir /path/to/index  \
	--readFilesIn <fq1> \
	--runThreadN <processorsN> \
	--outSAMtype SAM \
	--outFileNamePrefix /path/to/output
fi

--readFilesIn: input fastq files

Output

By default, STAR output following files

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- -rw-rw-r-- Aligned.out.sam
- -rw-rw-r-- Log.final.out
- -rw-rw-r-- Log.out
- -rw-rw-r-- Log.progress.out
- -rw-rw-r-- SJ.out.tab
- drwx------ _STARtmp

Aligned.out.sam holds the alignment results

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samtools view Aligned.out.sam |head -n1
A00582:731:HHGVJDSX2:1:1106:3586:16845  99      chr11   74699355        255     149M    =       74699383        24655   GTCAAGCTTATTTGATATAGTGGTATGTCCCTCCAGAAAAATCAAAAGTTGTGATCCCTGGATTTGAATTAAATATGCCACTATGTGGCTTCCACAGAGGGAAAAATGATTCTTTTTTTCAAGTGAATCCAATCAGCAACCAGTCAACA   FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF,FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF   NH:i:1 HI:i:1   AS:i:294        nM:i:0

Log.final.out records the alignment status

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cat Log.final.out
                                 Started job on |       Sep 15 17:26:21
                             Started mapping on |       Sep 15 17:27:00
                                    Finished on |       Sep 15 17:30:05
       Mapping speed, Million of reads per hour |       318.71

                          Number of input reads |       16378402
                      Average input read length |       288
                                    UNIQUE READS:
                   Uniquely mapped reads number |       14279182
                        Uniquely mapped reads % |       87.18%
                          Average mapped length |       287.27
                       Number of splices: Total |       14764233
            Number of splices: Annotated (sjdb) |       0
                       Number of splices: GT/AG |       14660617
                       Number of splices: GC/AG |       82087
                       Number of splices: AT/AC |       6046
               Number of splices: Non-canonical |       15483
                      Mismatch rate per base, % |       0.17%
                         Deletion rate per base |       0.01%
                        Deletion average length |       2.28
                        Insertion rate per base |       0.01%
                       Insertion average length |       1.86
                             MULTI-MAPPING READS:
        Number of reads mapped to multiple loci |       1337685
             % of reads mapped to multiple loci |       8.17%
        Number of reads mapped to too many loci |       54922
             % of reads mapped to too many loci |       0.34%
                                  UNMAPPED READS:
  Number of reads unmapped: too many mismatches |       0
       % of reads unmapped: too many mismatches |       0.00%
            Number of reads unmapped: too short |       342060
                 % of reads unmapped: too short |       2.09%
                Number of reads unmapped: other |       364553
                     % of reads unmapped: other |       2.23%
                                  CHIMERIC READS:
                       Number of chimeric reads |       0
                            % of chimeric reads |       0.00%

Ref:

https://github.com/alexdobin/STAR

完。