Hi I have a strand specific RNA-Seq experiment. The libraries were prepared based on dUTP and I was told to use forward strand.
I used Tophat to align the fastq files and merged replicates. I get a single bam file and used RseqQC on bam file. I get the following output:
This is PairEnd Data
Fraction of reads failed to determine: 0.0040
Fraction of reads explained by "1++,1--,2+-,2-+": 0.0087
Fraction of reads explained by "1+-,1-+,2++,2--": 0.9873
From RSeqQC documentation,:
1+-,1-+,2++,2–
read1 mapped to ‘+’ strand indicates parental gene on ‘-‘ strand
read1 mapped to ‘-‘ strand indicates parental gene on ‘+’ strand
read2 mapped to ‘+’ strand indicates parental gene on ‘+’ strand
read2 mapped to ‘-‘ strand indicates parental gene on ‘-‘ strand
I understood that the bam files, majority of reads, where read2 mapped to + strand with gene on + strand.
My question:
How do I set the option --library-type for cufflinks. Cufflinks offfers multiple options for library-type such as:
Supported library types:
ff-firststrand
ff-secondstrand
ff-unstranded
fr-firststrand
fr-secondstrand
fr-unstranded (default)
transfrags
in my case, based on rseq qc, shoudl i choose ff-secondstrand? or fr-firststrand or fr-reversestrand ?
appreciate your help.
Thanks
Adrian.
I used Tophat to align the fastq files and merged replicates. I get a single bam file and used RseqQC on bam file. I get the following output:
This is PairEnd Data
Fraction of reads failed to determine: 0.0040
Fraction of reads explained by "1++,1--,2+-,2-+": 0.0087
Fraction of reads explained by "1+-,1-+,2++,2--": 0.9873
From RSeqQC documentation,:
1+-,1-+,2++,2–
read1 mapped to ‘+’ strand indicates parental gene on ‘-‘ strand
read1 mapped to ‘-‘ strand indicates parental gene on ‘+’ strand
read2 mapped to ‘+’ strand indicates parental gene on ‘+’ strand
read2 mapped to ‘-‘ strand indicates parental gene on ‘-‘ strand
I understood that the bam files, majority of reads, where read2 mapped to + strand with gene on + strand.
My question:
How do I set the option --library-type for cufflinks. Cufflinks offfers multiple options for library-type such as:
Supported library types:
ff-firststrand
ff-secondstrand
ff-unstranded
fr-firststrand
fr-secondstrand
fr-unstranded (default)
transfrags
in my case, based on rseq qc, shoudl i choose ff-secondstrand? or fr-firststrand or fr-reversestrand ?
appreciate your help.
Thanks
Adrian.
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