FastQC per base sequence content

[analyst]

Does it seems acceptable (Global GC cotent for the species = 40%)

<original link removed>

[simonandrews]

Can you attach it to the forum? That site is blocked from here. Also it would be useful to see the per sequence GC plot as well as the per base plot.

[Bukowski]

Does it seems acceptable (Global GC cotent for the species = 40%)

<link removed>

Please do not use that host for images - it has NSFW content displayed prominently, and I have no desire to get sacked for viewing SeqAnswers links in an open plan office.

[analyst]

I understand and apologize. and thanks to the person moderating who posted image correctly

Can anyone please comment on the fastQC finding

[simonandrews]

I can't see an image reposted. The moderator only removed the original link. We still can't see your data.

[Orr Shomroni]

analyst, you may want to look at the following website:

http://www.bioinformatics.bbsrc.ac.uk/projects/fastqc/Help/

Introduction gives some general information on the FastQC algorithm. For specific information on the modules (what the developers consider as "pass", "warn" and "fail"), see under "Analysis modules" there will be some text files about each module and what their thresholds are.

This is a quote from the section on GC content:

Warning

This module issues a warning it the GC content of any base strays more than 5% from the mean GC content.

Failure

This module will fail if the GC content of any base strays more than 10% from the mean GC content.

Does this answer your question?

[pmiguel]

Looks acceptable to me.

What concerns you? If it is the low-level but periodic fluctuations in base levels across the read, that has been commented on before in another thread. There the period is three bases per cycle and more regular than what you see here.

--
Phillip

[analyst]

Thanks for the comments orr and phillip. Consider me beginner, just wanted to make sure the peaks at certain positions is nothing to worry about. It is more pronounced in the second picture I just posted, at 43. FastQC is warning me but I am not reading too much into it.

Simon, can you see the new picture I just posted, if not, I am also attaching here.

[pmiguel]

Okay, the new plot (the second one) is a different story. The G peak at 42 bases may indicate an issue of some sort.

--
Phillip

[stacy09]

help with fastQC per base sequence content

Hi,

I am a beginner of RNA-seq data analysis. I did fastQC of my samples and I found some variations at the beginning of reads for per base seqence content check. would you please tell me whether it is ok?

Many thanks in advance!

[kmcarr]

Hi,

I am a beginner of RNA-seq data analysis. I did fastQC of my samples and I found some variations at the beginning of reads for per base seqence content check. would you please tell me whether it is ok?

Many thanks in advance!

See this previous thread for a discussion of this phenomenon.

[stacy09]

kmcarr,

Thanks a lot!

[Aditi Verma]

Hello,

I ran FastQC through RNA-seq done on ribodepleted samples and the per base sequence content and per base GC content shows a very heavy bias (If I am correctly interpreting the results). Is this expected in data from ribodepleted RNA. Can this data be used at all?
Please find the image attached. Thanks for your help in advance!

[GenoMax]

@Aditi: Have you scanned/trimmed this data for presence of Illumina adapters? I wonder if you have a large percentage of adapter dimers (and no real inserts).

I recommend using bbduk.sh from BBMap for this purpose. Search for the thread on bbduk here.

[pmiguel]

Looks like about 20-25% of your reads are from adapter dimers.

--
Phillip