SEQanswers

Go Back   SEQanswers > Bioinformatics > Bioinformatics



Similar Threads
Thread Thread Starter Forum Replies Last Post
Illumina Hiseq 3000 / enough coverage for RNAseq analysis sheibani Illumina/Solexa 8 02-12-2016 04:46 AM
Lotsa new toys from Illumina: HiSeq X Five, 3000, 4000, NextSeq 550 GW_OK Illumina/Solexa 53 05-21-2015 12:30 AM
First HiSeq 3000 data DNATECH Illumina/Solexa 22 05-21-2015 12:24 AM
New rRNA removal kit for RNA-Seq epibio Vendor Forum 45 09-19-2013 07:38 AM

Reply
 
Thread Tools
Old 11-09-2017, 08:45 AM   #1
marnal
Junior Member
 
Location: Barcelona

Join Date: Nov 2017
Posts: 3
Default rRNA and adapter removal bondaries in RNA-seq HiSeq 3000 Illumina

Hello

I am new in RNA-seq analysis and it is difficult for me to find out info about quality assessment in the matter. I did an analysis with fastqc and MultiQC in a Paired End, 125nts (2x125), HiSeq3000 experiment. I obtained these two plots (attached) and I am not sure if a rRNA or adapter removal is required prior to alignment.

In the case of the adapter content plot it looks like all samples have adapters included, in fact none of the samples passed the QA. In the case of the plot with %GC content (I read it can be used to detect a possible rRNA enrichment) I see a proportion of reads having a GC content higher than 60%, but I am not sure if it is enough to confirm a high presence of rRNA that could affect to posterior analysis.

Any help will be useful!

Thanks

Magda
Attached Images
File Type: png fastqc_adapter_content_plot.png (112.4 KB, 4 views)
File Type: png fastqc_per_sequence_gc_content_plot.png (117.0 KB, 6 views)
marnal is offline   Reply With Quote
Old 11-09-2017, 10:10 AM   #2
GenoMax
Senior Member
 
Location: East Coast USA

Join Date: Feb 2008
Posts: 6,550
Default

You should scan/trim your sequences with a suitable program (bbduk.sh from BBMap, trimmomatic, cutadapt etc.). While some rely on aligners to soft-clip adapter sequences (and they likely will), trimming them beforehand ensures that your initial data is clean.

Presence of rRNA is a separate issue. If you feel that your data has rRNA reads in it then the best solution is to align a sample (or whole data) to rDNA repeat for your organism. While rRNA reads would not directly interfere with counts if you have uneven levels of them across samples then they can indirectly affect your results.
GenoMax is offline   Reply With Quote
Old 11-10-2017, 07:07 AM   #3
marnal
Junior Member
 
Location: Barcelona

Join Date: Nov 2017
Posts: 3
Default

Thanks for your quick answer, it was very useful! Just a last question, could you recommend me a tool or database I could use to make the alignment with the rDNA repeats of an organism?

Thanks
marnal is offline   Reply With Quote
Old 11-10-2017, 07:14 AM   #4
GenoMax
Senior Member
 
Location: East Coast USA

Join Date: Feb 2008
Posts: 6,550
Default

What organism are you working with?
GenoMax is offline   Reply With Quote
Old 11-10-2017, 08:05 AM   #5
marnal
Junior Member
 
Location: Barcelona

Join Date: Nov 2017
Posts: 3
Default

We are working with human
marnal is offline   Reply With Quote
Old 11-10-2017, 08:13 AM   #6
GenoMax
Senior Member
 
Location: East Coast USA

Join Date: Feb 2008
Posts: 6,550
Default

You can get the human rDNA repeat from here. If you do see contamination you can use bbsplit.sh from BBMap suite to separate those reads from rest of your data, if you wish.
GenoMax is offline   Reply With Quote
Reply

Tags
adapter contamination, adapter trimming, rrna contamination, rrna removal

Thread Tools

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off




All times are GMT -8. The time now is 11:25 AM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2017, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO