To identify which ‘--library-type’ setting to use with TopHat (PMID: 19289445, 23618408), Illumina specifically documents the types in the ‘RNA Sequencing Analysis with TopHat’ Booklet. For the TruSeq RNA Sample Prep Kit, the appropriate library type is 'fr-unstranded'. For TruSeq stranded sample prep kits, the library type is specified as 'fr-firststrand'. These post are also very informative: How to tell which library type to use (fr-firststrand or fr-secondstrand)? and How to determine if a library Is strand-specific. Another suggestion is to view aligned reads in IGV and determine the read orientation by one of two methods. First, you can have IGV color alignments according to strand using the ‘Color alignments’ by ‘First-of-pair strand’ setting. Second, to get more detailed information you can hover your cursor over a read aligned to an exon. ‘F2 R1’ means the second read in the pair aligns to the forward strand and the first read in the pair aligns to the reverse strand. For a positive DNA strand transcript (5' to 3') this would denote a fr-firststrand setting in TopHat, i.e. "the right-most end of the fragment (in transcript coordinates) is the first sequenced". For a negative DNA strand transcript (3' to 5') this would denote a fr-secondstrand setting in TopHat. ‘F1 R2’ means the first read in the pair aligns to the forward strand and the second read in the pair aligns to the reverse strand. See above for the complete definitions, but its simply the inverse for ‘F1 R2’ mapping. Anything other than FR orientation is not covered here and discussion with the individual responsible for library creation would be required. Typically ‘RF’ orientation is reserved for large-insert mate-pair libraries. Other orientations like ‘FF’ and ‘RR’ seem impossible with Illumina sequence technology and suggest structural variation between the sample and reference. Additional details are provided in the TopHat manual.
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