Question: How to tell is sequencing data is unstranded, firststrand, or secondstrand
0
gravatar for williamsbrian5064
6 months ago by
williamsbrian5064100 wrote:

Hi

I have some RNA-Seq data that I received from a colleague. I am trying to figure out if the data is unstranded, firststrand, or secondstrand. Is there an easy way to tell? I am running the data through salmon and this is what the lib_format_counts.json file is giving me. I think it is secondstranded?

read_files  "Sample1"
expected_format "SR"
compatible_fragment_ratio   0.9945605200682789
num_compatible_fragments    18794420
num_assigned_fragments  18897211
num_consistent_mappings 56858584
num_inconsistent_mappings   328289
MSF 0
OSF 0
ISF 0
MSR 0
OSR 0
ISR 0
SF  328289
SR  56858584
MU  0
OU  0
IU  0
U   0

Any ideas?

R rna-seq sequence assembly • 565 views
ADD COMMENTlink modified 6 months ago by swbarnes24.0k • written 6 months ago by williamsbrian5064100
2

Maybe ask the colleague?:)

ADD REPLYlink written 6 months ago by grant.hovhannisyan1.1k
2

What fun is that!

ADD REPLYlink written 6 months ago by Devon Ryan84k
1

Sometimes colleagues forget what they did or remember incorrectly. The data, on the other hand, doesn't lie.

ADD REPLYlink written 6 months ago by igor6.6k

haha I would be they haven't been the easiest to get in contact with. You know how it goes.

ADD REPLYlink written 6 months ago by williamsbrian5064100
1

There are some excellent answers posted already, but just in case you want to learn more about strandness, check this previous post: Read pair orientation : Illumina TruSeq Stranded mRNA library

ADD REPLYlink modified 6 months ago • written 6 months ago by igor6.6k

Great link! Very helpful info! I appreciate it!

ADD REPLYlink written 6 months ago by williamsbrian5064100
1
gravatar for Devon Ryan
6 months ago by
Devon Ryan84k
Freiburg, Germany
Devon Ryan84k wrote:

Yes, this appears to be the standard dUTP/secondstrand/ISR library. As an aside, this is the most likely library prep type these days.

ADD COMMENTlink written 6 months ago by Devon Ryan84k

Awesome! So the sequencing data is single end. Would this then be considered SR instead ISR?

ADD REPLYlink written 6 months ago by williamsbrian5064100
1

Yes, SR rather than ISR.

ADD REPLYlink written 6 months ago by Devon Ryan84k

Thanks you so much!

ADD REPLYlink written 6 months ago by williamsbrian5064100
1
gravatar for Bastien Hervé
6 months ago by
Bastien Hervé1.8k
Limoges, CBRS, France
Bastien Hervé1.8k wrote:

If you have no clue, take a look at RSeQC ( infer_experiment.py )

This program is used to “guess” how RNA-seq sequencing were configured, particulary how reads were stranded for strand-specific RNA-seq data, through comparing the “strandness of reads” with the “standness of transcripts”.

ADD COMMENTlink modified 6 months ago • written 6 months ago by Bastien Hervé1.8k
0
gravatar for swbarnes2
6 months ago by
swbarnes24.0k
United States
swbarnes24.0k wrote:

You can always eyeball it in IGV. It should be pretty easy to spotcheck half a dozen genes and see which way the reads go.

ADD COMMENTlink written 6 months ago by swbarnes24.0k
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