Is it possible to use genomic reads from PacBio and Nanopore with Illumina RNA-seq reads for de-novo assembly?

Question

[O.rka]

I have genomic reads from PacBio and Nanopore. I also have a few Illumina RNA-seq runs for this organism.

Is it possible to use ALL of these to generate a consensus assembly?

I know there is Hybrid-SPAdes but I don't know if you can give it both PacBio and Nanopore... plus, I don't think it wants RNA-seq reads.

[h.mon]

Genome or transcriptome assembly? You can't mix whole genome DNAseq and RNAseq for genome (or transcriptome) assembly, but you can use the RNAseq for scaffolding after genome assembly.

You may also use the Illumina RNAseq for polishing the Pacbio / NanoPore genome assembly.

I know there is Hybrid-SPAdes but I don't know if you can give it both PacBio and Nanopore

The SPAdes manual imply you can use both simultaneously. Why don't you try and find out?

[O.rka]

Yes, that is the terminology I was looking for with polishing my scaffolds with RNA-seq (thanks)! My goal is to have a polished genome assembly (not transcriptome assembly). I was under the impression that Hybrid-SPAdes wants shotgun genomic reads with long reads?

[harish]

How much coverage on the Longer reads are you looking at? Also how much data do you have from RNASeq?

It might just be easier to assemble the longer reads together and then use the RNASeq data to scaffold.

Answer 1

[h.mon]

I know at least Canu can use PacBio and NanoPore reads simultaneously to assemble a genome - there may be other assemblers out there capable of mixing PabBio and NanoPore input data.

After assembly, you may use Pilon with the RNAseq data to polish the draft assembly, see Using pilon with RNAseq data #50, Racon may also work.

[Shiyi]

Canu using long reads, then pilon using Illumina worked great for me.

Answer 2

[Buffo]

Is it possible to use ALL of these to generate a consensus assembly?

Besides possible, it is recommended since the high error rate of sequencing reads of PacBio and Nanopore comparing to illumina.

[O.rka]

Do you know if Hybrid SPAdes is design to take in RNA-seq data as well as the long read genomic data? Also, can it take both PacBio and Nanopore?

[h.mon]

Do you know if Hybrid SPAdes is design to take in RNA-seq data as well as the long read genomic data?

No, not together with shotgun genomic sequencing.

Also, can it take both PacBio and Nanopore?

Yes. From the manual:

To run SPAdes 3.12.0 you need at least one library of the following types:

    Illumina paired-end/high-quality mate-pairs/unpaired reads
    IonTorrent paired-end/high-quality mate-pairs/unpaired reads
    PacBio CCS reads

Illumina and IonTorrent libraries should not be assembled together. 
All other types of input data are compatible. SPAdes should not be used if
only PacBio CLR, Oxford Nanopore, Sanger reads or additional contigs are
available.