Favourite Software

iltisanni · 01-31-2018, 05:23 AM

Hello,

within the next months we want to start our own runs with the MinION.
___________________________
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?
___________________________

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 · 02-03-2018, 11:52 AM

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 · 02-04-2018, 09:12 PM

That's great.

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

gringer · 02-04-2018, 10:36 PM

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 · 02-05-2018, 12:50 AM

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

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01-31-2018, 05:23 AM	#1
iltisanni Junior Member Location: Mainz, Germany Join Date: Mar 2017 Posts: 9	Favourite Software Hello, within the next months we want to start our own runs with the MinION. ___________________________ 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? ___________________________ 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

02-04-2018, 10:36 PM	#4
gringer David Eccles (gringer) Location: Wellington, New Zealand Join Date: May 2011 Posts: 815	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. Last edited by gringer; 02-05-2018 at 01:04 AM.

02-03-2018, 11:52 AM	#2
gringer David Eccles (gringer) Location: Wellington, New Zealand Join Date: May 2011 Posts: 815	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.

02-04-2018, 09:12 PM	#3
iltisanni Junior Member Location: Mainz, Germany Join Date: Mar 2017 Posts: 9	That's great. Now how about the further software pipeline. Any recommendations for denovo Assembly, Alignment, (differential)gene-expression, snp/indel detection?

02-05-2018, 12:50 AM	#5
iltisanni Junior Member Location: Mainz, Germany Join Date: Mar 2017 Posts: 9	Thank you. I hope our MinION arrives this month, then I will use your recommended Software and see if the output satisfies me :-)