The need for DNAse I treatment depends on several factors, likeincluding:
The RNA extraction method: some procedures are more effective than others in removing gDNA (e.g., acidic phenol / chloroform extraction). It is therefore recommended to carefully evaluate the optimal RNA extraction method available for the specific sample type used as input.
The library prep preparation technology: the specific library prep method can also influence the impact of gDNA levels on the sequencing results.
3’ mRNA-Seq library preps (e.g., QuantSeq) are less affected by the presence of low levels of gDNA contamination than random primed whole transcriptome library preps.
Nevertheless, high gDNA levels
can also affect 3’ mRNA-Seq library preps and reduce the quality of the downstream data.
The sample type: some samples can have a higher DNA / RNA content and are therefore be prone to gDNA carry-over during retention through RNA extraction.
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Additional DNAse I treatment is recommended for samples that contain high levels of gDNA
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DNAse I treatment is always recommended for the following sample types:
Samples with high DNA / RNA content (e.g., like blood samples).
Low quality / FFPE samples.
Sample types Samples containing large quantities of short, extra-chromosomal DNASample undergoing .
Samples that have undergone mechanical disruption (due to fragmentation and increased gDNA fragmentation can increase carry-over of the gDNA potential)
What is the effect of gDNA contamination on my data?
The presence of gDNA contamination can strongly impact the sequencing data and lead to biases and quantification issues in the during data analysis.
This will also likely translate into the detection of a A typical signature of gDNA contamination includes:
A high proportion of intronic and intergenic reads and
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Loss of strand specificity (i.e., reads mapping to both strands)
For more information on gDNA contamination and DNAse treatment, please, check also our RNA LEXICON Chapter #5.