Single-end strand specific Rna-Seq: Which strand that the reads from.
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3 months ago
Chilly ▴ 10

Hi everyone,

I have searched online for a long time, but there is still no authoritative answer to this question (for single-end sequencing). So, I pointed it out here for clarification for the fresh people using single-end sequencing.

I am using TruSeq Stranded mRNA Sample Prep Kit (a strand-specific kit) to prepare my library. When I sequence (e.g. NovaSeq 6000 sequencing) this library:

  • For pair-end sequencing: the first read (read 1) is from the opposite strand (i.e. maps to the antisense strand), and the second read (read 2) is from the transcript strand (i.e. maps to the sense strand).
  • For single-end sequencing: all the reads are from the opposite strand (i.e. maps to the antisense strand).

The above info also works for Illumina Stranded mRNA, Illumina Stranded Total, and TruSeq Stranded Total kits, which are all RF/fr-firststrand stranded (dUTP) kits (includingTruSeq Stranded mRNA Kit).

Please feel free to correct me or add any more profound commands or suggestions here. Many thx.

mRNA Single-end strand-specific TruSeq • 307 views
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If you map the reads with STAR, it will provide you with some stats for reads mapped to +ive, -ive and either strand. That way you can diagnose the strandedness.

$ head
N_unmapped  1788830 1788830 1788830
N_multimapping  5204750 5204750 5204750
N_noFeature 12338116    33911209    12692401
N_ambiguous 1289974 11967   631457
ENSMUSG00000102693.2    0   0   0
ENSMUSG00000064842.3    0   0   0
ENSMUSG00000051951.6    51  0   51
ENSMUSG00000102851.2    0   0   0
ENSMUSG00000103377.2    12  0   12
ENSMUSG00000104017.2    6   0   6

From a previous post:

column 1: gene ID

column 2: counts for unstranded RNA-seq

column 3: counts for the 1st read strand aligned with RNA (htseq-count option -s yes)

column 4: counts for the 2nd read strand aligned with RNA (htseq-count option -s reverse)


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