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[iltisanni]

Hello,

within the next months we want to start our own runs with the MinION.
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One question besides:
First I read that the reads are in hast5 format and have to be extracted with eg. poretools.
Then I read that the output data is in fastq format.
What output format is generated by MinION-sequencing?
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Anyway, what I'm really interested in is what Software-pipelines you guys use and what you can recommend for denovo Assembly, Alignment, (differential)gene-expression, snp/indel detection?


I already found this list: http://bioinformaticsonline.com/blog...-data-analysis

[gringer]

Both the command-line program ONT provides for basecalling (Albacore) and the GUI Client that does sequencing and optionally basecalling (MinKNOW) can now output fastq files directly, no need to extract them from the fast5 files. The fast5 extraction was necessary about a year ago.

[iltisanni]

That's great.

Now how about the further software pipeline.
Any recommendations for denovo Assembly, Alignment, (differential)gene-expression, snp/indel detection?

[gringer]

I can tell you what I use, but it's unlikely to be what other people prefer, because there's a lot of manual computational legwork involved.

De-novo Assembly -- Canu. Flye and MARVEL sound promising, but I've yet to test them out.

Alignment -- minimap2 and/or LAST, depending on the application. LAST is particularly good at quickly picking up duplicated adapters in chimeric reads; I combine the output of LAST with my work-in-progress maf_bcsplit script to make results a bit easier to understand (it parses the MAF format produced by LAST into one-line-per match statistics like start/end/mapped percent).

Differential gene expression -- count transcripts directly via mapping with minimap2, use DESeq2 for differential expression tests. Kallisto/Sleuth may work as well, but possibly not given that I didn't have great results when using Salmon for gene counting based on minimap2-mapped transcripts.

SNP/INDEL detection -- I currently use samtools mpileup in combination with my own readstomper script to get base-level genotype frequencies. I'd like to look into doing a read-level haplotyping analysis, or at least multi-genotype (e.g. see this paper, or this paper), but have yet to try that. For mapping to known strains / genomes, Centrifuge should work; I think it's what ONT uses now for their What's In My Pot analysis tool.

[iltisanni]

Thank you.
I hope our MinION arrives this month, then I will use your recommended Software and see if the output satisfies me :-)

[iltisanni]

Update:

We sequenced some genomes so far (rapid sequencing Kit - SQK-RAD004) and the software we are using successfully on Ubuntu 18.04 is:

albacore - for basecalling (of course)
porechop - to trim adapters
canu - for assembly
nucmer - to find overlapping trimming points if the genome is circular
minimap2 - for alignment
samtools - for sorting and indexing the bam files from minimap2
nanopolish - for assembly polishing
pilon - for hybrid assembly polishing
Until now we haven't tried SNP/INDEL detection or differential gene expression.

So your recommended software works quite good and we are happy with it so far.

Thank You again !