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?
<p>Yes, the strand that&#39;s being mentioned is different in tophat/cufflinks than most other things. For tophat/cufflinks, it&#39;s the strand from cDNA construction that&#39;s being sequenced (i.e., either the first strand that&#39;s synthesized (fr-firststrand) or its reverse complement (fr-secondstrand)).</p>

<p>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.</p>
