How to get quality output for exome assembly ?

Question

[greyman]

Hi all I am very new in exome seqeucing for SNP variants calling. I got my overall alignment rate of 97.02%. Can Trimmomatic do exome trimming for Hiseq PE? i didnt have information about the sequencing cycle....

Also, from fastqc reports that my per base sequence quality is fine but the "per base sequence content" seems not okay...... http://oi64.tinypic.com/2nbrgjp.jpg

Last question, when i didnt put an index reference (-t) when i piped in my alignment into samtools view .... it looks fine for me so far, does anyone has any experience on this could give some comment to me? Thank you all very much :)

[Ram]

See: How to add images to a Biostars post

Your image address is http://oi64.tinypic.com/2nbrgjp.jpg, not http://tinypic.com/r/2nbrgjp/9

[greyman]

thank you, just change it

[WouterDeCoster]

Not correctly, read the post again.

[WouterDeCoster]

Why is your title about assembly?

Answer 1

[cschu181]

1. You can trim your exome reads with Trimmomatic just as you can do with WGS reads.
2. I wouldn't worry about the base sequence content of the first 10bp. That is the typical picture and in exome seq, which is more biased than WGS, it is even less alarming.
3. -t is not needed for viewing unless you don't have @SQ headers in your .bamfile. Maybe load your bam into a genome viewer, I find the sam-format to be a bit difficult to judge whether the alignments make sense.

[greyman]

thank you so much! I have no much information about adaptor sequence, only know that it generated by Hiseq sequencing. Would you think Trimmomatic using ILLUMINACLIP: Truseq gonna work? Would u recommend to try on BBDUK? Will input the BAM into genome viewer. There are @SQ in my BAM , how would you determine whether the alignment makes sense? I was using bowtie2

[cschu181]

Look at the FastQC report - if it shows Adapter contamination there (Adapter and Overrepresented Sequences) then you should run Trimmomatic using the default settings on their webpage and see what that does (run FastQC again on the trimmed reads). You might need to check which adapter file to use. Alternatively, you can of course use bbduk, just make sure to read the documentation there. I currently prefer bbduk due to it being a lot faster than Trimmomatic. Both are fine to use.

How would I determine whether the alignment makes sense... you could use bedtools to obtain information on which genes are covered (and how well, e.g. with what coverage) by your capture. If something is not covered well, you could check it in the viewer to assess the alignments in that region. There is a lot that you can do.