SEQanswers

Go Back   SEQanswers > Sequencing Technologies/Companies > Oxford Nanopore



Similar Threads
Thread Thread Starter Forum Replies Last Post
What's your favourite qPCR kit for Illumina library QC? ScottC Sample Prep / Library Generation 28 03-18-2016 03:08 AM
Add information and provenance to your favourite tools on the wiki dan Wiki Discussion 0 12-04-2015 01:33 AM
Looking for appropriate software lkral Bioinformatics 4 08-27-2014 10:37 AM
Commercial software help? vebaev Bioinformatics 1 06-19-2013 07:21 AM
population software lilimoon2010 Bioinformatics 7 02-25-2012 12:25 PM

Reply
 
Thread Tools
Old 01-31-2018, 05:23 AM   #1
iltisanni
Member
 
Location: Mainz, Germany

Join Date: Mar 2017
Posts: 21
Default 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
iltisanni is offline   Reply With Quote
Old 02-03-2018, 11:52 AM   #2
gringer
David Eccles (gringer)
 
Location: Wellington, New Zealand

Join Date: May 2011
Posts: 823
Default

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.
gringer is offline   Reply With Quote
Old 02-04-2018, 09:12 PM   #3
iltisanni
Member
 
Location: Mainz, Germany

Join Date: Mar 2017
Posts: 21
Default

That's great.

Now how about the further software pipeline.
Any recommendations for denovo Assembly, Alignment, (differential)gene-expression, snp/indel detection?
iltisanni is offline   Reply With Quote
Old 02-04-2018, 10:36 PM   #4
gringer
David Eccles (gringer)
 
Location: Wellington, New Zealand

Join Date: May 2011
Posts: 823
Default

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.
gringer is offline   Reply With Quote
Old 02-05-2018, 12:50 AM   #5
iltisanni
Member
 
Location: Mainz, Germany

Join Date: Mar 2017
Posts: 21
Default

Thank you.
I hope our MinION arrives this month, then I will use your recommended Software and see if the output satisfies me :-)
iltisanni is offline   Reply With Quote
Old 05-16-2018, 05:07 AM   #6
iltisanni
Member
 
Location: Mainz, Germany

Join Date: Mar 2017
Posts: 21
Thumbs up

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 !

Last edited by iltisanni; 07-18-2018 at 03:48 AM.
iltisanni is offline   Reply With Quote
Reply

Thread Tools

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off




All times are GMT -8. The time now is 04:57 AM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2018, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO