Hi all,
I have had a question the until now I could not find a clearly answer. I have some data for RNA seq that I have aligned. Observing the alignment done by STAR in IGV, I see things like this one:
My question here is: Which is the right direction of the transcript that produces the reads, knowing that the RNA-seq has been done in Single end?
I am little confused here because : If I look for the 3 first reads (at the top in blue) into my fastq file of RNA seq reads, these reads are:
@SRR.139896188
TCTGCAGACACCCTTCTCCGGCCGGAGCTG
+
AAAAAFF7FAFFFFFFFFFFFFFFFFFFAF
@SRR.107479696
TCTGCAGACACCCTTCTCCGGCCGGAGCTG
+
AAAA<FFAFAFFFFFFFFFFAFFFFFFFFF
@SRR.40743323
TCTGCAGACACCCTTCTCCGGCCGGAGCTG
+
AAAAAFF)FAFFFFFFFFFFFFFFFFFFFF
But in my alignment file (sam file) the reads look like this :
SRR.139896188 16 chr1 136042 255 30M * 0 0 CAGCTCCGGCCGGAGAAGGGTGTCTGCAGA FAFFFFFFFFFFFFFFFFFFAF7FFAAAAA NH:i:1 HI:i:1 AS:i:27 nM:i:1
SRR.107479696 16 chr1 136042 255 30M * 0 0 CAGCTCCGGCCGGAGAAGGGTGTCTGCAGA FFFFFFFFFAFFFFFFFFFFAFAFF<AAAA NH:i:1 HI:i:1 AS:i:27 nM:i:1
SRR.40743323 16 chr1 136042 255 30M * 0 0 CAGCTCCGGCCGGAGAAGGGTGTCTGCAGA FFFFFFFFFFFFFFFFFFFFAF)FFAAAAA NH:i:1 HI:i:1 AS:i:27 nM:i:1
So, the reverse complement of these reads is aligned in the genome Is this meaning that the direction of the transcript is the reverse sens of the genome ?
For these blue reads, here the preparation library protocol used:
Fragment polyadenylation, Linker ligation to do after reverse transcription, Then circularization to deplete ribosomal RNA, Next PCR- Amplification and Sequencing - single layout on NextSeq 500
What if I look again in the image : the red reads are aligned in the same sens as they are in the fastq (RNA reads). So, I might say that the transcript direction is the actual direction of the genome showed in the image?
For these red reads, here the preparation library protocol used:
cDNA synthesis and library generation performed according to TruSeq Stranded Total RNA Sample Preparation protocol (Illumina). mRNA-seq libraries was subjected to sequencing on a NextSeq 500 instrument (Illumina) to yield 75 bp single-end reads.
I hope to be clear enough to have a clear answer. :)
Thanks in advance.
How to add images to a Biostars post
Is your RNA-seq from a stranded or unstranded protocol/library prep?
There are two strategies used :
For the first set (blue reads) the preparation correspond to Ribo-seq protocol which is:
The second set for the red reads the protocol is :
Thank you about how to add images here. :)
I'm not sure about the ribo-seq reads, but TruSeq stranded reads are in the opposite direction of transcription.
Thank you very much for your answer. I figured it out: as you said TruSeq stranded reads are in the opposite direction of transcription whereas ribosome-seq reads will be in the direction of transcription.