Illumina hiseq and pacbio isoseq data on same set of samples- combined analysis help?

Question

[datanerd]

Hi all,

I have data generated on the same set of samples using both illumina (RNAseq ribozero) and pacBio isoseq method. Was wondering if anyone has experience in combined analysis of these data sets or if there is any paper out there that utilizes both the datasets.

Thanks!

Mamta

[Adrian Pelin]

Is this DNA or RNA sequencing? What do you want to do? Are you interested in de-novo assembly algorithms?

[User 59]

The Iso-Seq in the title suggests they are asking about RNA as it's a transcriptomics library prep method for PacBio

[datanerd]

As Daniel pounted out, it is RNAseq data generated using Ribozero illumina and Isoseq pacbio methods. Thanks Daniel.

I want to utilize the benefits of having both datasets in isoform identification, DEG analysis and studying lncRNA.

[annashadutta18]

Hi Mamta,

I need to perform Illumina Truseq and pacbio seq on the same set of sample. Could you let me know more about the sample preparation method you used.

Thanks Annasha Dutta

Answer 1

[User 59]

I bookmarked a paper last week that you might find interesting: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3864310/

Although transcriptional and posttranscriptional events are detected in RNA-Seq data from second-generation sequencing, full-length mRNA isoforms are not captured. On the other hand, third-generation sequencing, which yields much longer reads, has current limitations of lower raw accuracy and throughput. Here, we combine second-generation sequencing and third-generation sequencing with a custom-designed method for isoform identification and quantification to generate a high-confidence isoform dataset for human embryonic stem cells (hESCs). We report 8,084 RefSeq-annotated isoforms detected as full-length and an additional 5,459 isoforms predicted through statistical inference. Over one-third of these are novel isoforms, including 273 RNAs from gene loci that have not previously been identified. Further characterization of the novel loci indicates that a subset is expressed in pluripotent cells but not in diverse fetal and adult tissues; moreover, their reduced expression perturbs the network of pluripotency-associated genes. Results suggest that gene identification, even in well-characterized human cell lines and tissues, is likely far from complete.

[datanerd]

Thanks Daniel. This seems something like I would be interested in doing too.

What would you suggest regarding doing DEG study? Use the other dataset as validation or combine dataset or any other way people might have done it?

Thanks so much for your time.

Mamta