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Running STAR aligner with paired-end and single-end reads simultaneously

Hi all

So, I recently got some RNA-seq raw reads, both paired end (2 x 150 bp) and single-end (1x75 bp) I want to map them using STAR aligner. My main questions are then, how would you deal with these? Can STAR take both paired-end and single-end .fq files simultaneosuly? Or mapping separetely and then merging the bam files is also possible?

Any ideas?

Thank you for your advice

star rna-seq paired-end single-end
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written by nanoide nanoide

Thank you all for your thoughts and useful comments!

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written by nanoide nanoide

Thank you all for your responses.

Please allow to ask for a couple of clarifications: * I get STAR cannot deal with paired-end sequencing and single-end sequencing of different length at the same time. Do you know any aligner that can? * Do you know of any published paper that have done something similar to what you suggested?

Thank you very much

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written by nanoide nanoide

BBMap suite has a tool that allows bbmap.sh to be used for single and paired-end reads in the same alignment job.

$ bbwrap.sh in1=read1.fq,singletons.fq in2=read2.fq,null out=mapped.sam append
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written by GenoMax GenoMax

You have not said if this is the same library sequenced two different ways. That will also have implications on how you do the data analysis.

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written by GenoMax GenoMax

Hi, thanks for your responses. This is indeed the same library sequenced two different ways. I'll check out BBmap suite, thanks

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written by nanoide nanoide

3 answers

I don't think STAR can take them both together. I'd process the two separately, and merge results at the end if it looks like the two experiments are telling you the same thing.

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written by swbarnes2 swbarnes2

And I would suggest to maybe hold on to merging the results until you look at a PCA of the data first to ensure there is not a batch effect of sequencing types etc...

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written by lshepard lshepard

Thank you both for your answers. So I guess I will map separately. Then with the bam files I will use deeptools plotPCA (maybe plotCorrelation too?) to check and then samtools merge. Does that sound good? Any thoughts?

Thanks!

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written by nanoide nanoide

Hi, please let me ask you for a clarification. When you stated 'merge the results at the end if it looks like the two experiments are telling you the same thing', did you mean merging the output from STAR (i.e. bam files) or counting also independently and then suming the counts if they are correlated, cluster together... etc Thank you!

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written by nanoide nanoide

Depends on how you are normalizing reads. If you are just generating raw counts, you could just combine the counts. Otherwise, you should probably merge the bams, recalculate the counts and renormalize.

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written by swbarnes2 swbarnes2

Ok, thank you very much

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written by nanoide nanoide

I typically use single-end 50 bp reads for gene expression analysis.

If you are just interested in getting counts for differential expression (and FPKM/CPM for visualization), perhaps trim the longer R1 from the PE experiment to 75 bp?

To be safe, I probably would start by processing them separately and seeing how well the replicates cluster. If they really look like technical replicates, I think you could justify combined analysis with the trimmed reads in your Supplemental Materials.

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written by Charles Warden Charles Warden

Hello,

I would also recommend producing a correlation (Spearman or Pearson) distance matrix to see how well the samples correlate within their group. DESEQ2 has this option to produce heatmaps of distance matrix as well.

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written by Ashastry Ashastry

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