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ENCODE RNA-seq (paired-end)

ID: 1020F6D9-C921-7B9D-11AB-89FF40B13ECB

This pipeline uses STAR for mapping reads and RSEM for quantifying the expression levels of genes and isoforms.

Input files

Two gzip-compressed files, for each read (InputFile1 for R1 and InputFile2 for R2) and a chromosome size file (InputFile3)

Parameters

  1. RndSeed: random seed for RSEM, by default 12345
  2. ForwardProb: forward probability for RSEM. 1 for the forward design, 0 for the reverse design. By default 0. For libraries generated with the dUTP method, this value should be 0. If you are not sure about the design, you can probe it with infer_experiment.py. If most of the reads can be explained by:
    1. "1+-,1-+,2++,2--", then set this value as 0;
    2. "1++,1--,2+-,2-+", then set this value as 1.
  3. NCPUs: CPU cores available for mapping and expression quantification.
  4. ramGB: Max buffer size, by default 8.
  5. RndSeed: Random seed for RSEM, by default 12345.

Environment

  • Operation System: Linux/Unix
  • CPU cores: 8
  • Memory: 32GB

Steps

Software Parameter
STAR --genomeDir {{StarIndexDir}} --readFilesIn {{InputFile:1}} {{InputFile:2}} --readFilesCommand zcat --runThreadN {{NCPUs||4}} --genomeLoad NoSharedMemory --outFilterMultimapNmax 20 --alignSJoverhangMin 8 --alignSJDBoverhangMin 1 --outFilterMismatchNmax 999 --outFilterMismatchNoverReadLmax 0.04 --alignIntronMin 20 --alignIntronMax 1000000 --alignMatesGapMax 1000000 --outSAMheaderCommentFile COfile.txt --outSAMheaderHD @HD VN:1.4 SO:coordinate --outSAMunmapped Within --outFilterType BySJout --outSAMattributes NH HI AS NM MD --outSAMtype BAM SortedByCoordinate --quantMode TranscriptomeSAM --sjdbScore 1
mv Aligned.sortedByCoord.out.bam {{JobName}}_genome.bam
rsem-sam-validator Aligned.toTranscriptome.out.bam
mv Aligned.toTranscriptome.out.bam {{JobName}}_anno.bam
samtools flagstat {{JobName}}_genome.bam > {{JobName}}_genome_flagstat.txt
samtools flagstat {{JobName}}_anno.bam > {{JobName}}_anno_flagstat.txt
samtools quickcheck {{JobName}}_genome.bam
samtools quickcheck {{JobName}}_anno.bam
STAR --runMode inputAlignmentsFromBAM --inputBAMfile {{JobName}}_genome.bam --outWigType bedGraph --outWigStrand Stranded --outWigReferencesPrefix chr
bedSort Signal.UniqueMultiple.str1.out.bg Signal.UniqueMultiple.str1.out.bg
bedGraphToBigWig Signal.UniqueMultiple.str1.out.bg {{InputFile:3}} {{JobName}}_minusAll.bw
bedSort Signal.Unique.str1.out.bg Signal.Unique.str1.out.bg
bedGraphToBigWig Signal.Unique.str1.out.bg {{InputFile:3}} {{JobName}}_minusUniq.bw
bedSort Signal.Unique.str2.out.bg Signal.Unique.str2.out.bg
bedGraphToBigWig Signal.Unique.str2.out.bg {{InputFile:3}} {{JobName}}_plusAll.bw
bedSort Signal.UniqueMultiple.str2.out.bg Signal.UniqueMultiple.str2.out.bg
bedGraphToBigWig Signal.UniqueMultiple.str2.out.bg {{InputFile:3}} {{JobName}}_plusUniq.bw
rsem-calculate-expression --alignments --estimate-rspd --calc-ci --seed {{RndSeed||12345}} -p {{NCPUs||4}} --no-bam-output --ci-memory {{ramGB||8}}000 --forward-prob {{ForwardProb||0}} --paired-end {{JobName}}_anno.bam {{RsemIndex}} {{JobName}}_rsem

References

Reference Description
StarIndexDir

STAR index for the genome.
RsemIndex Path to the folder storing the index for RSEM. The path also needs to end with `reference_name`. For example, /path/to/the/folder/rsem
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