Can I align paired end reads and orphan reads to reference sequences at the same time

Question

[Kurban]

Hi everyone,

I have clipped my raw reads to get rid of poor quality reads by using The Nesoni clip tool and I got clipped_R1.fq, clipped_R2.fq and clipped_single.fq (orphan reads) files. Here clipped_R1.fq and clipped_R2.fq are paired, but clipped_single.fq is single reads. Now I wanna align all these reads to reference sequences and then get FPKM value. so i used bowtie2:

bowtie2 [options]* -x <bt2-idx> {-1 <m1> -2 <m2> | -U <r>} -S [<hit>]

(Usage from the manual)

I wrote this:

bowtie2 -a -X 800 -p 3 -x ~/Desktop/RNA-seq/CD/all_animals_transcription_factors_nt \
    {-1 ~/Desktop/RNA-seq/CD/nesoni_clip_clead_reads/clipped_R1.fq -2 ~/Desktop/RNA-seq/CD/nesoni_clip_clead_reads/clipped_R2.fq | -U ~/Desktop/RNA-seq/CD/nesoni_clip_clead_reads/clipped_single.fq} | samtools view -Sb - > hits.bam

but that did not work.

Can I do that alignment that paired end and orphan reads at the same time to reference sequences? If I could, how? Could anyone give me some suggestions?

Answer 1

[GouthamAtla]

This should work. post the exact error if any. Hope clipped_R1.fq and clipped_R2.fq are in proper order.

bowtie2 \
  -a \
  -X 800 \
  -p 3 \
  -x ~/Desktop/RNA-seq/CD/all_animals_transcription_factors_nt \
  -1 ~/Desktop/RNA-seq/CD/nesoni_clip_clead_reads/clipped_R1.fq \
  -2 ~/Desktop/RNA-seq/CD/nesoni_clip_clead_reads/clipped_R2.fq \
  -U ~/Desktop/RNA-seq/CD/nesoni_clip_clead_reads/clipped_single.fq | \
    samtools view -Sb - > hits.bam

[Kurban]

Hey GouthamAtla ,

Yeah, it works if I deleted this part from the command:

-U ~/Desktop/RNA-seq/CD/nesoni_clip_clead_reads/clipped_single.fq

But I do not wanna leave out the clipped_single.fq, single reads(this reads leaving unpaired after trimming the low quality paired end reads). I wanna align them too.

[joaopaulo_bio]

But if you deleted the "-U" part from your command how do you expect to make bowtie use the unpaired reads?