Polishing PACBIO corrected assembly using ONT and ILLUMINA data (.fastq)

Question

[K]

Hi,

I have corrected PACBIO assembly of a plant genome (query) and would like to polish the assembly with ONT reads and Illumina (target.fastq). I am using Minimap2 but it gave me .sam and .paf output like below.

minimap2 -ax map-ont ref.fa ont-reads.fq > aln.sam (I am not sure where the polishing has been done. How can see if the gaps has been filled?)

minimap2 -a ref.fa ont-reads.fq > aln.paf (This give me target coordinates that I want to extract the sequence for to see if there is any overlapping between PACBIO/ONT/ILLUMINA.

Any suggestion is welcome?

Thank you

[samuel.a.odonnell]

You want to look at genome polishing tools such as racon and medaka for ONT and pilon for illumina.

Just to note, your ONT reads are hopefully longer than your pacbio reads so you want to use them to assemble the genome structure too.

[K]

Thank you. I am using RACON for polishing HIFIASM assembled contigs by adding ONT. I will considere pilon for illumina polishing

so, basically I am following

PACBIO -> ONT -> ILLUMINA

ONT -> PACBIO -> ILLUMINA (polish and error correction at each step). Does it sound ok?.

Which tools do you recommend to assemble ONT assembly? Is Hifiasm, IPA would work?

Much appreciated!!!

[samuel.a.odonnell]

Hifiasm I believe is designed purely for pacbio reads. You lay want to look at Canu or Raven or numerous other assemblers now available.

But again, swapping for an ONT assembly would only be beneficial if your read length, particularly your read N50, is greater than for your pacbio data