Thanks for replying and clarifying a few things.
Just so I'm straight, I can use debarcode to add the UMIs from fastq read file #1 into
the read header of fastq read file #2.
I would then align to a genome to generate a bam, then use retag to switch the UMI from
the read header to the RX position in the bam - then I'd be able to use picard to
dedup with the RX barcode_tag. Feasibly, once the UMI is added to the header, I could
still use the current je-markdupes to dedupe (it being a fork of picard)?
Previously, I've been using fgbio, but this involved aligning the data in Galaxy,
exporting it and annotating with UMI's and then loading back in. Ideally, we'd
like to keep the entire workflow within Galaxy (hence the original question).
From: Charles Girardot <charles.girardot(a)embl.de
March 2018 22:25:57
To: Brooks, Tony
Cc: galaxy-dev(a)lists.galaxyproject.org; Jelle Scholtalbers
Subject: Re: [galaxy-dev] Best practices for utilising Unique Molecular Indexing
please see inline answers
On 27. Mar 2018, at 16:45, Brooks, Tony <a.brooks(a)ucl.ac.uk>
Thanks for replying. Yes, I'd definitely be interested in the command line version.
we’ll try to push the version we have in conda tomorrow.
I'm assuming I can use je retag to add the UMI from a separate
read to my sample fastq (already demultiplexed by bcl2fastq) then use the de-markdupes
already in Galaxy to dedupe? Would this work?
I think you got me wrong (or I got you wrong!): je retag lets you transfer UMI/BARCODE
information embedded in the read name of a *bam* file to proper bam BC/RX/QX/OX/BZ/MI
tags. Indeed many tools (will) expect this info to be available in BAM tags and not in
The current version of je markedupes expects the UMI info in the read name. We havent
updated je markedupes to get this info from BAM tags yet (even in je-suite2, not fully
ready as mentioned before)
Btw, I did find a small bug in markdupes. On the NextSeq, the fastq
header contains a space, e.g.
When I run markdupes, I get an error as it thinks the UMI is "1043", not
read names in BAM files can’t have spaces so the read name in your BAM is likely to be :
and by default, the UMI is expected in the last position ; hence '1043'
Please double check how the read names look in your bam file.
> From: Charles Girardot <charles.girardot(a)embl.de
Sent: 26 March 2018 12:05:22
> To: Brooks, Tony
> Cc: galaxy-dev(a)lists.galaxyproject.org
> Subject: Re: [galaxy-dev] Best practices for utilising Unique Molecular Indexing
> Hi Tony,
> sounds like you need is the Je-Suite version 2 which we
will release soon-ish.
> In this version, the new “je debarcode” module allows you
to define an unlimited number of FASTQ input together with their layout (describing
BARCODE, UMI and SAMPLE positions). One can define an unlimited number of output FASTQ
layouts by combining the BARCODE, UMI and SAMPLE slots defined in input layouts (one slot
can be written in multiple layouts if need be). For example, you can output the UMI in
their own separate file instead of/in addition to keeping them in the read header.
> This version also let you keep the demultiplexed reads in
a single output file (for single end I mean), map this file to the genome then use the new
“je retag” to transfer the BARCOD/EUMI info embedded in read header to proper BAM tags.
This should make it easier to deal with single-cell datasets i.e. manipulate a unique file
instead of hundreds/thousands…
> The command line version is close to completion (“je
retag” needs a bit more testing) and I believe we could push this in conda rather quickly.
The bad news is we haven’t started to write/update the Galaxy wrappers yet.
> Let me know if you are interested to try the command line
On 26. Mar 2018, at 11:45, Brooks, Tony <a.brooks(a)ucl.ac.uk> wrote:
> > Hi
> > We are currently seeing a number of methods that are utilising the power of
unique molecular indexing. Unfortunately, there is no consensus on how libraries should be
configured, and therefore no consensus for how to deal with them within Galaxy.
> > Often libraries that have the UMI placed directly
downstream of the first (i7) index, such as ones using the IDT xGen adapter set
Sometimes UMI’s exist in place of the second (i5) index
> > In both cases, the recommended workflows are
convoluted and all the necessary tools do not currently exist in the toolshed (so that the
datasets need to be taken out of galaxy, processed and reloaded).
> > It is possible to use bcl2fastq to output the UMI as an additional fastq file,
but this would then require me to create a dataset triplicate (not pair) which afaik we
can’t do (yet).
> > A quick Google/toolshed search had me find UMI-Tools
& Je-Suite which both exist in Galaxy.
> > Both these tools assume the UMI is “in-line” (i.e. at the beginning of the read
1 or read2 – not its own read), extract/remove the UMI and place it in the read header,
where it is then used further down the line to dedup the bam file.
> > Does anyone know of any tools that would take the UMI
from a separate fastq and use it to tag the headers of actual read data. Or alternatively,
a tool that will paste the UMI tag onto the 5’ end of the read fastq? And whether these
steps can be done within Galaxy, or maybe prior to fastq upload?
> > Anyone have a method/workflow for UMI’s?
> > Thanks in advance
> > Tony
> > ___________________________________________________________
> > Please keep all replies on the list by using "reply all"
> > in your mail client. To manage your subscriptions to this
> > and other Galaxy lists, please use the interface at:
> > https://lists.galaxyproject.org/
> > To search Galaxy mailing lists use the unified search
> > http://galaxyproject.org/search/
> Charles Girardot
> Head of Genome Biology Computational Support (GBCS)
> and Senior Bioinformatician in the Furlong Lab
> European Molecular Biology Laboratory
> Tel: +49 6221 387 -8585
> Fax: +49-(0)6221-387-8166
> Email: charles.girardot(a)embl.de
> Skype: charles_girardot
> Web : http://gbcs.embl.de
> Room V205
> Meyerhofstraße 1,
> 69117 Heidelberg, Germany
Head of Genome Biology Computational Support (GBCS)
and Senior Bioinformatician in the Furlong Lab
European Molecular Biology Laboratory
Tel: +49 6221 387 -8585
Web : http://gbcs.embl.de
69117 Heidelberg, Germany