my mouse RIP-seq data have abnormal GC content and the fastqc result in 'GC content' have 2 peaks, first one is about 55% in x axis, y axis is about 500000. the second one as about 85% in axis,y axis is about 1600000, very sharp peak.besides, the GC content each base is not about 25% for A,T,G,C. they looks like electrocardiogram from 0bp to 150 bp. I checks the unmaapped reads in my bam files and blast with NCBI nr database, which shows that these reads were come from mouse. I think it isn't the contaminate with other sequence, I really can't find how this can be?
2 answers
Hi,
since you have RIPseq data, your libraries don't follow the assumptions of a FastQC. You amplify a limited set of sequences which have not the diversity as e.g. in whole genome sequencing data. You may have a look at the "common reason for warning" section of FastQC'S documentation (e.g. here)
On the other hand, you may check if the sequencer was a NextSeq machine. In 2 colour chemistry, you may discover poly-G sequences; see here for more information.
Cheers,
Michael
Log in to answer this question.
-
What is the importance of checking per base sequence content and per sequence GC content during Qua…
written by VBer
-
Per sequence GC content failed in Human RNA-seq Data
written by tahseen.abbas •
-
FastQC results with abnormal GC content and Overrepresented sequences in RIP-seq datasets
written by yoyostudents5775 •
-
How to interpret and trim this plots?
written by eyonesi •
-
fastqc gc-content peak and adapter content
written by David
-
Identifying contaminant sources
written by rachanaj •
-
FastQC report about WGBS.
written by hxlei613
-
RNA Seq: GC Content extra peak
written by firestar
-
Bimodal GC content
written by Folder40g
-
Understanding Fastqc Output- Please Help
written by Kash
There are 2 replicates sample in my RIP-seq for each condition, but another sample performed better than this. I maped my reads to mm10 with hisat2, the overall mapping rate about 69%,and nearly 50% of total reads aligned concordantly exactly 1 time. sequencer was illumina Hiseq 3000, And I am wondering whether this is caused by RNA degradation or PCR bias?
Please use
ADD COMMENT/ADD REPLYwhen responding to existing posts to keep threads logically organized.This comment should have gone under @michael's answer.