Trimmomatic -> STAR

Question

[xd_d]

Hello everyone,

R1: forward R2: reserve

DATA: RNA-Seq data

i used trimmomatic for paired-end reads. I get 4 outputs. sample.R1.trimmed.fastq, sample.R2.trimmed.fastq, sample.R1.unpaired.fastq , sample.R2.unpaired.fastq,

Now i want align these files with STAR-Aligner against the reference genome hg38. Should i run STAR without unpaired reads ? or should I run with the unpaired output and run STAR for the unpaired reads as single-end data ?

Thanks

[Carlo Yague]

I usually align using only the paired reads. In my datasets, there are very few orphan reads (<< 1%) and its not worth the trouble (in my opinion) to incorporate them. But I would be interested to hear a better argumented answer.

[jake.hagen]

I don't trim reads when using star, as I believe it includes soft trimming when aligning reads. Is there a special reason you are using hard trimming? or and argument for using trimmomatic? Thanks

[GenoMax]

While you can get away with aligners "soft clipping" data as needed it is perhaps safe to have your data scanned/trimmed using a proper trimming program. That way you have a "clean" data file that can be used for any downstream application (including assembly where presence of adapters would be problematic).

[xd_d]

I want to make a quality trimming and Trimmomatics is a flexible trimmer. I read this Paper https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4103590/ and I thought why not to improve my reads before I use Star. hmm

[GenoMax]

That is perfectly fine.

Question is do you have enough reads in the "unpaired" files to bother using them as @carlo said above.

[xd_d]

28825048 reads forward and reserve 27798716 (96,44%) Forward only 728334 (2,53%) Reserve only 261697 (0.91 %) dropped 36301 ( 0.13%)

I run : java -jar trimmomatic-0.35.jar PE -phred33 input_forward.fq.gz input_reverse.fq.gz output_forward_paired.fq.gz output_forward_unpaired.fq.gz output_reverse_paired.fq.gz output_reverse_unpaired.fq.gz ILLUMINACLIP:TruSeq3-PE.fa:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36

i think i takte only forwand and reserve for star