Removing barcode and adapters?

Question

[manekineko]

Hi,

I have pair-end files, what is the normal step to proceed remove the adapters and barcode seq or the other way around? I probably will use FASTX toolkit if it is suitable?

@HWI-ST1075L:227:S1FDTG6ACXX:8:1101:1329:2093 1:N:0:ACAGTG
AAGCCGATCAGATACCTGGAATTCTCGGGTGCCAAGGAANTNNAGNNACNCAGTGANCTCNTATGCCGTNTTCTGCTTGAAAAAAAAAAAAAAAAAAAAAA
++

Is the analysis should be done in file pairs R1 R2 or?

Answer 1

[GouthamAtla]

First use FastQC tool to check the quality of the data. If you see any drop in the quality at the begining or at the end of the reads, trim the reads. FastQC has also shows over represented sequences. If you see any adapters listed there, use tools like cutadapt/TrimGalore to remove the adapter sequences.

For a good quality RNA-Seq or DNA-Seq data, there is no need to remove the adapters. There is very less likely that the adapter contamination will be there. Its mandatory for microRNA data. In general, it depends on the fragment size that goes in to the sequencer.

Answer 2

[manekineko]

It is miRNA data, thats why I'm confused as the seq are so long and pair-end, usualy I analysed single end as the miRs are quite short

[GouthamAtla]

Its strange to see 100bp PE data for miRNA. You should definitely remove the adapters then.

[manekineko]

and the Barcode probably after that?

The strange question is after I clean the R1 and R2 what to do if it is pair-end if it is miRNA? what is actually the data on the R2?!

[michael.ante]

Do you have a dual indexed sample? If so, I would go for fastx' barcode splitter.

Otherwise, you'll find the barcode in the read-name:

@HWI-ST1075L:227:S1FDTG6ACXX:8:1101:1329:2093 1:N:0:ACAGTG
                                                    ^^^^^^
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[Picasa]

What you highlight is rather the illumina index than the barcode right ?