How to interpret the difference among these three options in strandedness from HTSeq-count

Question: How to interpret the difference among these three options in strandedness from HTSeq-count

4.1 years ago by

United States

nalandaatmi • 70 wrote:

Dear All,

I am interested in calculating the % of reads associated to globin gene and rRNA genes. Right now, I am not sure whether my paired end RNAseq data has followed strand specific protocol or not. I requested the incharge person to inform me.

Meanwhile, I selected all the three options for strandedness (no,yes,reverse) in htseq-count.

How do I get the strand (sense,antisense) information

How to interpret the Stranded:Reverse counts

Globin genes	Stranded:No	Stranded:Yes	Stranded:Reverse
HBB	40204	40197	7
HBA1	38811	38795	16
HBA2	129847	129770	77
HBG1	1566	1566	0
HBG2	2750	2750	0
HBD	3	3	0
HBE1	1	0	1
HBZ	0	0	0
HBQ1	9	3	6
MB	4	0	4
CYGB	294	2	354
NGB	289	2	319

How to interpret the difference among these three options

Stats from special counters

Special counters	Stranded:No	Stranded:Yes	Stranded:Reverse
__no_feature	56289350	94180089	56914563
__ambiguous	625347	18161	343824
__too_low_aQual	0	0	0
__not_aligned	0	0	0
__alignment_not_unique	30631662	30631662	30631662

rna-seq sequence next-gen alignment • 1.7k views

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modified 4.0 years ago by Alternative • 230 • written 4.1 years ago by nalandaatmi • 70

4.0 years ago by

Alternative • 230

Alternative • 230 wrote:

Check the following explanation:

http://onetipperday.blogspot.de/2012/07/how-to-tell-which-library-type-to-use.html

Also, RSeqQC have a script that counts the different cases and tells you what type of stranded library you have. Check their infer_experiment.py script (I think I tried it long time ago but don't remember how accurate it is but should be ok).

http://rseqc.sourceforge.net/#infer-experiment-py

ADD COMMENT • link written 4.0 years ago by Alternative • 230

4.1 years ago by

Biomonika (Noolean) ♦ 3.1k

State College, PA, USA

Biomonika (Noolean) ♦ 3.1k wrote:

Take your file with mapped reads (bam or sam) and open it in IGV. Then right click and choose coloring by first-in-pair read strand. If all genes are colored with the same color (either pink or blue), your protocol is strand-specific. Each gene will be colored based on the fact if the transcription is sense or antisense compared to the reference. If your protocol is not strand-specific, you will se mix of both colors.

ADD COMMENT • link written 4.1 years ago by Biomonika (Noolean) ♦ 3.1k

4.1 years ago by

Alternative • 230

Alternative • 230 wrote:

I always like to see how things look like on IGV. I recommend loading the tracks and check as Noolean proposed but additionally, I would take a couple of small transcripts (with few reads) and check if the counts on IGV match with those of HTSeq.

Also, as Antonio said too, the person that generated the library has to give this information.

ADD COMMENT • link written 4.1 years ago by Alternative • 230

4.1 years ago by

Antonio R. Franco • 4.1k

Spain. Universidad de Córdoba

Antonio R. Franco • 4.1k wrote:

One will know when your library has been constructed stranded or not.. You must purchase and use specific reagents and follow a determined protocol

Don't you have that information ?

If you know that your library is stranded, you should give that information to your mapping program and to HTSeq-Count as well

ADD COMMENT • link written 4.1 years ago by Antonio R. Franco • 4.1k

Dear Noolean/Pierre/Antonio, I received the information now. The RNASeq library has been constructed based on strand specific protocol.

Sure, I will check the bam file using IGV and validate the counts provided by htseq-count.

But to get bam files, I used tophat for alignment step and it produced the accepted_hits.bam file. Using the following command for tophat

tophat -p 6 -o $outdir $bowtie_index $fastq_r1 $fastq_r2

I didn't mention any library-type. But I got the information now that my RNAseq is based on strand specific protocol.

Which below option should I need to select?

I believe, that now I should rerun my tophat with strand information as Antonio mentioned.

Below content from following link (https://ccb.jhu.edu/software/tophat/manual.shtml#toph):

--library-type

The default is unstranded (fr-unstranded). If either fr-firststrand or fr-secondstrand is specified, every read alignment will have an XS attribute tag as explained below. Consider supplying library type options below to select the correct RNA-seq protocol.

Library Type	Examples	Description
fr-unstranded	Standard Illumina	Reads from the left-most end of the fragment (in transcript coordinates) map to the transcript strand, and the right-most end maps to the opposite strand.
fr-firststrand	dUTP, NSR, NNSR	Same as above except we enforce the rule that the right-most end of the fragment (in transcript coordinates) is the first sequenced (or only sequenced for single-end reads). Equivalently, it is assumed that only the strand generated during first strand synthesis is sequenced.
fr-secondstrand	Ligation, Standard SOLiD	Same as above except we enforce the rule that the left-most end of the fragment (in transcript coordinates) is the first sequenced (or only sequenced for single-end reads). Equivalently, it is assumed that only the strand generated during second strand synthesis is sequenced.

ADD REPLY • link written 4.1 years ago by nalandaatmi • 70

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