How do I know if I have DNA contamination in RNA-seq data?

Question

[blur]

Hi,

I want to know whether I have DNA contamination in my RNA-seq data. I have a library with DNAse and one without DNAse and another pair with additional treatment [DNAse(+) vs. DNAse(-), DNAse(+)+X vs. DNAse(-)+X]

I tried:

1) Checking overall alignment rate of DNAse(+) vs. DNAse(-) and DNAse(+)+X vs. DNAse(-)+X] It is slightly higher on DNAse free samples

2) Checking alignment vs. transcripts It's all over the place - DNAse(+)+X has lowest rate, then DNAse(-) (without treatment), then DNAse(+) (without treatment) and finally the least background was in DNAse(-)+X... But I figured that might be due to rRNA? If it had not been sufficiently cleaned in some libraries it would not map to transcripts.

3) Looking at the reads in IGV after alignment to see if there is background/baseline in non-exon areas.

I am out of ideas, anyone know what else I can do?

Thanks!

[Fabio Marroni]

Basically, you have tried almost everything. I would suggest a massive effort on step 3.

For example you might count the number of reads that align over introns in all your samples. The higher the number of such reads the higher the DNA contamination (assuming that the proportion of pre-mRNA in your samples is the same. I guess it was total RNA (not poly-A selection).

I also guess you didn't remove rRNA.

I am not sure, but maybe filtering rRNA reads might give you better estimates.

[blur]

Thanks, I'll try it all (the library is after experimental rRNA depletion, but I guess it never hurts to check again)

[Kevin Blighe]

Was this a sample mis-labeling, by any chance?

[cpad0112]

1. Build intronic sequences fasta
2. Index it
3. Use fastqscreen
4. You should see only small % mapping to the test reads. blur

[zebasilio]

Hi!

I am trying to check for possible DNA contamination on the fastq files. Therefore I am using this tool, read_distribution: http://rseqc.sourceforge.net/#read-distribution-py

I then check for the percentage of reads aligned to intronic tags to check if there is DNA contamination.
Now I do not know how high must be the percentage of reads aligned to the intronic region to be considered DNA contamination.

Could you please help me in this regard?

Best, José

[ATpoint]

How high is it? As often there is no fixed cutoff.

[zebasilio]

Hi!

I am trying to check for possible DNA contamination on the fastq files. Therefore I am using this tool, read_distribution: http://rseqc.sourceforge.net/#read-distribution-py

I then check for the percentage of reads aligned to intronic tags to check if there is DNA contamination.
Now I do not know how high must be the percentage of reads aligned to the intronic region to be considered DNA contamination.

Could you please help me in this regard?

Best, José

[GenoMax]

Please don't post the same question twice. You should also not use SUBMIT ANSWER box to ask additional questions in an existing thread.

ATpoint asked you to provide some results/data. Unless you do that we can't help you since he has already indicated that there is no fixed cutoff.

Answer 1

[michael.ante]

Hi blur,

Try RSeQC's read_distribution.py. With DNA-contamination, you'll observe more introns and more intergenic 'tags'.

Cheers,

Michael