consistent problem of strand-specific information between TopHat/RSeQC/picard

Question

[pengchy]

The RNAseq strand-specific library was constructed using Illumina's strand specific kit: TruSeq stranded sample prep kits, which is based on dUTP method. As the documentation on illumina (http://www.illumina.com/documents/products/technotes/RNASeqAnalysisTopHat.pdf), the library type should be "fr-firststrand". I have mapped the data using TopHat with library type "fr-firststrand". And then I check the output bam file using picard "CollectRnaSeqMetrics.jar " and "RSeQC-2.6.1/scripts/infer_experiment.py", which give me the results listed below:

First from picard: The first column is from: STRAND=SECOND_READ_TRANSCRIPTION_STRAND, and the second column is from: STRAND=FIRST_READ_TRANSCRIPTION_STRAND. It is surprised me that the SECOND_READ_TRANSCRIPTION_STRAND give more CORRECT_STRAND_READS, just contrary to my expectation.

                                SECOND_READ_TRANSCRIPTION_STRAND    FIRST_READ_TRANSCRIPTION_STRAND
CORRECT_STRAND_READS            14040054                            138566
INCORRECT_STRAND_READS          138566                              14040054

Second from RSeQC:

This is PairEnd Data Fraction of reads failed to determine: 0.0000
Fraction of reads explained by "1++,1--,2+-,2-+": 0.0446
Fraction of reads explained by "1+-,1-+,2++,2--": 0.9554

It seems that picard using different meaning of the first and second as TopHat and Cufflinks, isn't it?

Answer 1

[Devon Ryan]

Yes, the strand that's being mentioned is different in tophat/cufflinks than most other things. For tophat/cufflinks, it's the strand from cDNA construction that's being sequenced (i.e., either the first strand that's synthesized (fr-firststrand) or its reverse complement (fr-secondstrand)).

Almost everything else is talking about the DNA strand to which a read/pair aligns. I personally tend to think in terms of this rather than in the steps of library construction, but to each their own.

[pengchy]

Hi Devon Ryan, according to your explanation, the picard's strand information is from DNA, so their strand information are contrary to each other. It make sense. Thank you.