SEQanswers

Go Back   SEQanswers > Applications Forums > RNA Sequencing



Similar Threads
Thread Thread Starter Forum Replies Last Post
FastQC,kmer content, per base sequence content: is this good enough mgg Bioinformatics 10 11-06-2013 11:45 PM
FastQC Kmer content: spike in reverse read reubennowell Bioinformatics 4 06-12-2013 03:56 AM
FastQC: odd kmer content zshuhua Introductions 3 05-13-2013 08:36 PM
kmer content warning in FastQC vallejov RNA Sequencing 0 04-05-2013 11:10 AM
weird kmer-content peak in RNA-seq data kareldegendt Bioinformatics 2 08-22-2012 04:45 PM

Reply
 
Thread Tools
Old 06-09-2013, 06:24 PM   #1
Ann7
Junior Member
 
Location: NZ

Join Date: Nov 2012
Posts: 1
Default FastQC: 3' bias (weird peaks) in Kmer content graphics

Hi All,

My samples were run on Illumina HiSeq2000 using paired-ended stranded RNA-Seq protocol and the output reads are 100bp. The quality of my data are not bad in general. But I got some weird peaks at 3' end in FastQC kmer content graphics (plz see the attached graphs) and most of peaks are "GAAGA". Since this is part of the Illumina adapter (GATCGGAAGAGCTCGTATG), I tried Cutadapt (Cutadapt -a GATCGGA inputfile outputfile) to trim all the adapters at 3' end. This did get rid of the peaks in some of my samples, but the peaks still retain in a few samples, and another kmer "GAGGA" poped up. So I'm sure whether the 3' bias were caused by adapter remnant.

I also has a question about the 5' end bias. I was told they were caused by random hexmars. But shouldn't the hexmars only appear at 5' end of Read1? Why Read2 has the same bias at 5' end?

I will use trimmomatic to trim and filter the data next. But I'm not sure whether I should trim off the 3' end of Read2 or not, as the average overlapping between Read1 and Read2 is only 21bp (the average size of the library was 300bp, but all the fragments larger than 200bp were selected, which means the average inserts were of 179bp).

I would appreciate it very much if anyone has a better clue of what happened at both ends of the reads in my data set. Many thanks!

Attached graphs:
1: kmer content graph of Read1 (raw data)
2: kmer content graph of Read2 (raw data)
3: kmer content graph of Read1 (after Cutadapt)
4: kmer content graph of Read2 (after Cutadapt)
5: quality graph of Read2 (raw data)

The Cutadapt reports of Read1 and Read2:
52F-R1: Adapter 'GATCGGA', length 7, was trimmed 713126 times.
Processed reads: 5456168
Processed bases: 545616800 bp (545.6 Mbp)
Trimmed reads: 713126 (13.1%)
Trimmed bases: 9418910 bp (9.4 Mbp) (1.73% of total)
52F-R2: Adapter 'GATCGGA', length 7, was trimmed 544449 times.
Processed reads: 5456168
Processed bases: 545616800 bp (545.6 Mbp)
Trimmed reads: 544449 (10.0%)
Trimmed bases: 7082484 bp (7.1 Mbp) (1.30% of total)
Attached Images
File Type: png R1_raw_kmer.png (92.4 KB, 72 views)
File Type: png R2_raw_kmer.png (77.8 KB, 44 views)
File Type: png R1_cutadapt_kmer.png (93.4 KB, 32 views)
File Type: png R2_cutadapt_kmer.png (88.1 KB, 25 views)
File Type: png R2_raw_quality.png (17.4 KB, 35 views)
Ann7 is offline   Reply With Quote
Old 06-19-2013, 06:14 AM   #2
syfo
Just a member
 
Location: Southern EU

Join Date: Nov 2012
Posts: 103
Default

I would try with the full adapter sequence in the Cutadapt command line ("GATCGGAAGAGCTCGTATG") in case some similarity score threshold was not reached.
syfo is offline   Reply With Quote
Old 06-19-2013, 06:31 AM   #3
mastal
Senior Member
 
Location: uk

Join Date: Mar 2009
Posts: 666
Default

When you are trimming adapters from the 3' end, you have to trim the same number of bases from the 3' end of both R1 and R2.

Basically, if you are reading into the adapter sequences, it means your insert is shorter than the read length, and the whole insert has been sequenced twice. The sequences of R1 and R2 will be reverse complements of each other, apart from the 3' end where you read into the adapters.
mastal is offline   Reply With Quote
Reply

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 10:50 PM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2021, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO