How to normalize long-read RNA-seq data for comparison with short-reads
0
0
Entering edit mode
Bernardo • 0
@27b596fa
Last seen 1 hour ago
United States

I am working on a project comparing RNAseq quantification results between Illumina short-reads and Nanopore long-reads and I have a couple questions about comparing the quantification results from these two technologies. More specifically I need some help with figuring out how to normalize the data for the comparisons within samples and between samples. So far I have come up with the following plan:

  1. Using CPM to compare gene/transcript expression within each sample sequenced with nanopore. For example, comparing if gene.X transcripts are more abundant than gene.Y transcripts within sample_1 sequenced with nanopore.

    • Using CPM instead of TPM for nanopore seems like a good option since our nanopore runs do not have transcript length bias. Does this sound like a good strategy?
  2. Using TPM to compare gene/transcript expression within each sample sequenced with illumina. For example, comparing if gene.X transcripts are more abundant than gene.Y transcripts within sample_1 sequenced with illumina.

    • Using TPM instead of CPM for illumina seems like a good option since illumina has transcript length bias (a single long transcript will have more counts that a single short transcript). Does this sound like a good strategy?
  3. Here is where I am having trouble coming up with a good normalization strategy. Comparing gene/transcript expression between the same sample sequenced with illumina and nanopore. e.g., performing a spearman correlation between gene expression in sample_1 sequenced with illumina and sample_1 sequenced with nanopore. I am not sure what would work here since Illumina has transcript length bias and nanopore does not. Do you have any suggestions?

Any help here will be greatly appreciated.

Best, Bernardo

NanoporeRNASeq LongRead Normalization ShortRead IlluminaRNASeq • 81 views
ADD COMMENT
1
Entering edit mode

I suggest to post that over at biostars.org to get a broader audience of long-read people.

ADD REPLY
0
Entering edit mode

Just did that, thank you for the suggestion.

ADD REPLY
0
Entering edit mode

Discussion for this question continuing on Biostars: https://www.biostars.org/p/9552419/

ADD REPLY

Login before adding your answer.

Traffic: 466 users visited in the last hour
Help About
FAQ
Access RSS
API
Stats

Use of this site constitutes acceptance of our User Agreement and Privacy Policy.

Powered by the version 2.3.6