Question: low assignment reads % in RNA-seq
gravatar for parinv
13 days ago by
pune, India
parinv30 wrote:

I performed RNA-seq analysis on 8 samples(2 control and 6 samples) . I used featureCounts function (Rsubread package) to count the number of reads. I found that Number of assigned reads were very poor. I am attaching the table here for your reference. I used following function:

fc<- featureCounts(annot$Sample,isPairedEnd=TRUE, GTF.attrType = "transcript_id", annot.ext = "GCF_000203855.3_ASM20385v3_genomic.gtf", nthreads = 3, isGTFAnnotationFile = TRUE)

I do not understand why assignment% is low, when alignment % is found higher. Can anyone help me think of possible reasons for why I'm getting such low values for assignment?

Thanks a lot. Parin

rna-seq R • 123 views
ADD COMMENTlink modified 11 days ago by sysboolean70 • written 13 days ago by parinv30

check if multi-mapped reads are counted or not and use correct strandedness. Talk to the core and check if ribodepletion worked for all the samples.

ADD REPLYlink modified 10 days ago • written 11 days ago by cpad011215k

can you post the alignment summary metrics for example like this

ADD REPLYlink written 10 days ago by lakhujanivijay5.4k
gravatar for solomoncharles77
13 days ago by
solomoncharles7750 wrote:

One possible reason is that you may need to indicate the strand information of your library. Besides, this issue has been discussed in multiple threads, like here and here. Those threads and others should contain suggestions that can help you resolve the issue. Good luck.

ADD COMMENTlink written 13 days ago by solomoncharles7750
gravatar for sysboolean
11 days ago by
sysboolean70 wrote:

Can you post more information about the origin of the samples ? Someone I know once performed RNA-seq on what they thought was human cell culture, but got poor alignment percents like yours. Further investigation (BLASTing a few reads against nr db) showed that the cells were from a non-human mammal. Might help to run Fastqscreen to make sure your samples were not switched during sequencing.

ADD COMMENTlink modified 11 days ago • written 11 days ago by sysboolean70
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