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 methods available, as they may also vary 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.
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Nevertheless, high gDNA levels
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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 therefore be are more prone to gDNA carry-over during RNA extraction.
When RNA samples contain high levels of gDNA, a DNAse treatment is recommended.
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DNAse I treatment is always recommended for the following sample types:Difficult samples, like blood samples (
o Samples with high DNA / RNA content (e.g., blood samples).
o Low quality / FFPE samples.
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o Samples containing large quantities of short, extra-chromosomal DNA
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.
o Samples that have undergone mechanical disruption (
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gDNA fragmentation can increase carry-over
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The presence of gDNA contamination can strongly impact the sequencing data and lead to biases and quantification issues in the data analysis.
This will also likely translate into the detection of a high proportion of intronic and intergenic reads and loss of strand specificity.
potential)
ATTENTION! DNase I should be removed before library preparation as it may affect the efficiency of the library generation.
NOTE: For more information on gDNA contamination and DNAse treatment, please, check also our RNA LEXICON Chapter #5.
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