Per base sequence content error

[barthez95]

Hi,

I have 3 questions related with my fails in FastQC report.


1. I attached a printscreen presents graph with per base sequence content. Could tell me why I see deviation in the 3' end. Should I cut 3 bp on the 3' end?

2. My report shows red flag in Per tile sequence quality. I see few red tiles on the heatmap. What I should do with it?

3. I would like to use BWA-MEM to do alignment to reference genome. I heard that BWA-MEM expect min. length of read = 70 bp. Should I set MINLEN in Trimmomatic = 70?

[GenoMax]

Fails (red X) does not automatically mean your data is bad. You need to take into account context of what kind of experiment this is. Can you tell us about that?

1. Aligners should soft-clip (remove from alignment) bases that do not map, so those at the end of the reads should be taken care of.
2. Do nothing specific. You don't have a high number of bad tiles.
3. 70 is pretty high. What genome is this data from. Even for human genome reads > 40 bp will map fine.

[barthez95]

Thank you. I do not know the context. I have just obtained this raw data and I would like to do SNP calling.

So in 1 and 2 cases I can just ignore without trimming?

3. I read that BWA-MAM want min length = 70 (in manual). It is bovine genome (Bos taurus).

[GenoMax]

Context was the type of experiment. Sounds like this is genome resequencing.

If you want to reduce possibility of reads multi-mapping by chance then going with longer reads is better. Minlen of 70 should be strict enough.

You would want to scan and trim your data so any extraneous sequence present (adapter etc.) can be removed. I recommend using "bbduk.sh" from BBMap suite (thread here as well as a guide). Set "minlen=70".

[barthez95]

Ah, ok I understand. It is WGS sequencing

[barthez95]

Hmm you recommend a BBDUK trimmer. What do you think about trimmomatic? I would like to use with this set of settings: TruSeq3-PE.fa:2:30:10 SLIDINGWINDOW:4:12 MINLEN:70

My supervisor prefers Trimmomatic. Can I use it with settings above?

Thank you!

[GenoMax]

Sure you can use trimmomatic. The options looks right (I don't know them off the top of my head) and should work.