Dear all,
We'll have our Geuvadis RNAseq analysis group call again today
(Thursday) at 2pm.
Things to be discussed:
- update on sequencing progress
- miRNA QC issues
- Tuuli will present some slides on how genetic variants affect
mapability, and ways to correct for it
- mRNA and miRNA data processing pipelines
Call details:
Spain
902 125 136
Other countries
+34 91 495 18 99
Participants' Pin-code:
764989*
best regards,
Tuuli
--
Tuuli Lappalainen, PhD
Department of Genetic Medicine and Development
University of Geneva Medical School
CMU / Rue Michel-Servet 1
1211 Geneva 4
Switzerland
Tel. +41-(0)22-3795550
tuuli.lappalainen(a)unige.ch
Hello,
We've found a slight discrepancy in the Geuvadis RNAseq lab ID number info.
This is due to my mistake: In the RNAseq protocol text in November
there's a table of lab IDs, but then I forgot about this and assigned
partly different IDs when sending out the sample information sheets.
This caused some confusion now that we've actually had data to exchange.
Sorry about this, but I'm glad we caught it early (thanks Peter,
Matthias, Olof).
From now on, please use the following IDs that correspond to the sample
information sheets. I also enclosed the sequencing protocol with an
updated table.
Because of this error, in the miRNAseq QC slides from Matthias the
samples labeled as Leiden are actually from Uppsala.
have a nice weekend,
Tuuli
Correct Lab # # in the previous protocol table
UNIGE (Geneva) 1 1
CRG (Barcelona) 2 2
MPIMG (Berlin) 3 3
ICMB (Kiel) 4 4
HMGU (Munich) 5 7
UU (Uppsala) 6 5
LUMC (Leiden) 7 6
--
Tuuli Lappalainen, PhD
Department of Genetic Medicine and Development
University of Geneva Medical School
CMU / Rue Michel-Servet 1
1211 Geneva 4
Switzerland
Tel. +41-(0)22-3795550
tuuli.lappalainen(a)unige.ch
Hello,
Here are miRNA QC results of the replicate samples from Matthias.
best regards,
Tuuli
Tuuli Lappalainen, PhD
Department of Genetic Medicine and Development
University of Geneva Medical School
CMU / Rue Michel-Servet 1
1211 Geneva 4
Switzerland
Tel. +41-(0)22-3795550
tuuli.lappalainen(a)unige.ch
-------- Original Message --------
Subject: Fwd: Re: [Geuvadis_rna_analysis] Geuvadis RNAseq analysis
group TC - slides
Date: Thu, 23 Feb 2012 16:28:23 +0100
From: Matthias Barann <m.barann(a)ikmb.uni-kiel.de>
To: Tuuli Lappalainen <Tuuli.Lappalainen(a)unige.ch>
Hello Tuuli,
could you please try to forward the attached pdf file to the others. I
tried it in three different ways (as excel sheet, zip file and pdf) but
always get an error.
Thanks,
Matthias
-------- Original Message --------
Subject: Re: [Geuvadis_rna_analysis] Geuvadis RNAseq analysis group TC
- slides
Date: Thu, 23 Feb 2012 16:26:39 +0100
From: Matthias Barann <m.barann(a)ikmb.uni-kiel.de>
To: geuvadis_rna_analysis(a)lists.crg.es
Dear all,
here is the quality analysis we have done on the common miRNA samples.
There's one sample which shows a weird read-length for the genome
mapping (NA20527, page 11-12). However, in the miRNA mapping it's not
that much different anymore, but it misses some more miRNAs (page 6).
And there are a few samples which show almost equal amounts of short
reads (14 bp) compared to the number of 22 bp reads.
Everything else looks quite similar.
One other thing I noticed is, that the sample NA19095 seems to contain a
(very little) bit more poly-A tagged RNAs (page 2, upper graph). This is
probably due to the extraction, as all institutes have roughly the same
amount of polyA-trimmed reads for the same samples.
For mapping only reads were used, which had the adapter successfully
removed.
best wishes,
Matthias
--
Matthias Barann
Institute of Clinical Molecular Biology
Christian Albrechts University Kiel
Schittenhelmstr. 12
D-24105 Kiel, Germany
m.barann(a)ikmb.uni-kiel.de
+49 - (0)431 - 597 8681 (office)
Dear all,
Here is the Geuvadis RNAseq draft for the abstract for the Biology of
Genomes meeting. Please *comment and give your author and affiliation
info by Tuesday Feb 21*.
Regarding the author list: The length limitation includes author &
affiliation info, so we need to keep it very short for this abstract. We
suggest the following:
- analysis people with major contribution: Tuuli Lappalainen,
Michael Sammeth, Natalja Kurbatova, Tim Strom, Peter t' Hoen, Matthias
Barann, Marc Sultan, Esther Lizano, Manuel Rivas. If someone else feels
that they should be included, we can try to squeeze in an additional
name or two.
- One PI per institute - *Kiel and Berlin, please indicate who*:
Alvis Brazma, Ann-Christine Syvanen, Thomas Meitinger, Gert-Jan van
Ommen, Philip Rosenstiel */* Stefan Schreiber, Ralf Sudbrak */* Hans
Lehrach, Ivo Gut, Xavier Estivill, Emmanouil T Dermitzakis
- and last: "on behalf of the Geuvadis Consortium"
*Everyone on the author list: please reply to this email with your name
and affiliation info. *Due to length limitations, please include only
your Geuvadis partner affiliation and use abbreviations when possible.
best regards,
Tuuli
Author information example:
First name: Tuuli
Middle initial:
Last name: Lappalainen
Email: tuuli.lappalainen(a)unige.ch
Affiliation name: University of Geneva
Department: Dept of Genetic Medicine and Development
Street: R. Michel Servet 1
City : Geneva
Postal code: 1211 4
Country: Switzerland
--
Tuuli Lappalainen, PhD
Department of Genetic Medicine and Development
University of Geneva Medical School
CMU / Rue Michel-Servet 1
1211 Geneva 4
Switzerland
Tel. +41-(0)22-3795550
tuuli.lappalainen(a)unige.ch
Hello,
So, I'd like to ask all the labs that have miRNA data to upload 3 fastq
files to the ftp site.
I just uploaded our files, and I'll be expecting files from Barcelona
(Marta/Esther/Sergi), Berlin (Marc), Kiel (Matthias). Does Munich have
miRNA data? Please do this asap.
Detailed instructions:
- upload to
ftp-private.ebi.ac.uk/upload/geuvadis/wp4_rnaseq/main_project/working/miRNA_QC
. User geuvadis, password k2vP3kjJ .
- to make the file processing easy, please name the fastq files
according to the naming instructions:
SAMPLE_ID.SeqLabNumber.MI_YYMMDD_Lane_1.fastq.gz . The lab numbers are:
Geneva=1, Barcelona=2, Berlin=3, Kiel=4, Munich=5. E.g. one of our files
is HG00096.1.MI_120104_4_1.fastq.gz
- choose 3 random files, or pick ones that have a little bit different
numbers of total reads
- upload also a readme file with at least your name and email and the
read length
- use raw fastq files without any adaptor trimming, or if you want, you
can trim all the reads to 36 bp
thanks,
Tuuli
--
Tuuli Lappalainen, PhD
Department of Genetic Medicine and Development
University of Geneva Medical School
CMU / Rue Michel-Servet 1
1211 Geneva 4
Switzerland
Tel. +41-(0)22-3795550
tuuli.lappalainen(a)unige.ch
Dear all,
here is our analysis of the miRNA samples of 4 institutes (UNIGE, MPIMG,
ICMB, CNAG, see attatched pdf file).
This is basically what I did:
1. trim reads to 36 bp (prinseq).
2. remove adapter sequence (cutadapt), split into trimmed reads and
untrimmed reads
3. remove polyA tails (cutadapt)
4. map trimmed reads against genome and miRNA reference (the one from
the FTP server).
5. count reads per miRNA
There are larger differences considering the amount of adapter sequences
within the samples and the library diversity.
best wishes,
Matthias
--
Matthias Barann
Institute of Clinical Molecular Biology
Christian Albrechts University Kiel
Schittenhelmstr. 12
D-24105 Kiel, Germany
m.barann(a)ikmb.uni-kiel.de
+49 - (0)431 - 597 8681 (office)
Hello all,
Please find the Geuvadis RNAseq TC minutes enclosed.
Action items:
-All the labs with miRNA data (everyone except Leiden, Uppsala?) should
upload 3 fastq files (preferably of different read counts; before any
trimming) to the ftp site, to
/upload/geuvadis/wp4_rnaseq/main_project/working/miRNA_QC. Then everyone
should analyze these files using their standard methods to make sure
that the data from different labs is of comparable quality. These
analyses need to be done asap.
-UNIGE will finish the details of invoicing the cell & RNA costs
-Tuuli will write and send around an abstract for Biology of Genomes
best regards,
Tuuli
--
Tuuli Lappalainen, PhD
Department of Genetic Medicine and Development
University of Geneva Medical School
CMU / Rue Michel-Servet 1
1211 Geneva 4
Switzerland
Tel. +41-(0)22-3795550
tuuli.lappalainen(a)unige.ch
Tuuli Lappalainen, PhD
Department of Genetic Medicine and Development
University of Geneva Medical School
CMU / Rue Michel-Servet 1
1211 Geneva 4
Switzerland
Tel. +41-(0)22-3795550
tuuli.lappalainen(a)unige.ch
-------- Original Message --------
Subject: RE: Rut, it seems that they cannot connect to the call can you
double check ? I´m in a meeting...
Date: Tue, 14 Feb 2012 16:09:07 +0100
From: Rut Carbonell Garcia <rut.carbonell(a)crg.eu>
To: Esther Lizano <Esther.Lizano(a)crg.eu>, Gabrielle Anne Bertier
<gabrielle.bertier(a)crg.eu>
CC: Tuuli Lappalainen <Tuuli.Lappalainen(a)unige.ch>
Try it now please
*De:*Esther Lizano
*Enviado el:* martes, 14 de febrero de 2012 16:06
*Para:* Gabrielle Anne Bertier
*CC:* Tuuli Lappalainen; Rut Carbonell Garcia
*Asunto:* Re: Rut, it seems that they cannot connect to the call can you
double check ? I´m in a meeting...
the problem is the pin code, it says invalid. could we use the
coordinator pin?
On 2/14/12 4:04 PM, Gabrielle Anne Bertier wrote:
*_14th of February at 4-5pm, 15 ppl_*
Spain
902 125 136
Other countries
+34 91 495 18 99
Participants' Pin-code:
764989*
*From:*geuvadis_rnaseq-bounces@lists.crg.es
<mailto:geuvadis_rnaseq-bounces@lists.crg.es>
[mailto:geuvadis_rnaseq-bounces@lists.crg.es] *On Behalf Of *Tuuli
Lappalainen
*Sent:* Tuesday, February 14, 2012 4:02 PM
*Cc:* geuvadis_rnaseq(a)lists.crg.es
<mailto:geuvadis_rnaseq@lists.crg.es>;
geuvadis_rna_analysis(a)lists.crg.es
<mailto:geuvadis_rna_analysis@lists.crg.es>
*Subject:* Re: [Geuvadis_rnaseq] [Geuvadis_rna_analysis] Geuvadis RNAseq
TC today at 4pm
These were the codes that I got from Gabrielle, I hope that she can fix
this!
Tuuli
Tuuli Lappalainen, PhD
Department of Genetic Medicine and Development
University of Geneva Medical School
CMU / Rue Michel-Servet 1
1211 Geneva 4
Switzerland
Tel. +41-(0)22-3795550
tuuli.lappalainen(a)unige.ch <mailto:tuuli.lappalainen@unige.ch>
On 2/14/12 4:01 PM, M.Sammeth wrote:
invalid code 764989*
On Tue, Feb 14, 2012 at 3:46 PM, Tuuli Lappalainen
<Tuuli.Lappalainen(a)unige.ch <mailto:Tuuli.Lappalainen@unige.ch>> wrote:
Sorry - here's the attachment.
Tuuli Lappalainen, PhD
Department of Genetic Medicine and Development
University of Geneva Medical School
CMU / Rue Michel-Servet 1
1211 Geneva 4
Switzerland
Tel.+41-(0)22-3795550 <tel:%2B41-%280%2922-3795550>
tuuli.lappalainen(a)unige.ch <mailto:tuuli.lappalainen@unige.ch>
On 2/14/12 3:45 PM, Tuuli Lappalainen wrote:
Dear all,
Here is a slide of small RNA quality analysis to discuss on the call.
best regards,
Tuuli
Tuuli Lappalainen, PhD
Department of Genetic Medicine and Development
University of Geneva Medical School
CMU / Rue Michel-Servet 1
1211 Geneva 4
Switzerland
Tel.+41-(0)22-3795550 <tel:%2B41-%280%2922-3795550>
tuuli.lappalainen(a)unige.ch <mailto:tuuli.lappalainen@unige.ch>
On 2/14/12 12:37 PM, Tuuli Lappalainen wrote:
Here are the call details
Spain
902 125 136
Other countries
+34 91 495 18 99
Participants' Pin-code:
764989*
best regards,
Tuuli
Tuuli Lappalainen, PhD
Department of Genetic Medicine and Development
University of Geneva Medical School
CMU / Rue Michel-Servet 1
1211 Geneva 4
Switzerland
Tel.+41-(0)22-3795550 <tel:%2B41-%280%2922-3795550>
tuuli.lappalainen(a)unige.ch <mailto:tuuli.lappalainen@unige.ch>
On 2/14/12 9:18 AM, Tuuli Lappalainen wrote:
Dear all,
As a reminder, we have a Geuvadis RNAseq TC today (14th) at 4 PM.
On the agenda:
- update on sequencing progress
- miRNA quality
- RNAseq meetings
April 16-17 in Geneva, future meetings?
- cell line & RNA extraction costs
- abstract for Biology of Genomes meeting at Cold Spring Harbor
- collaborations
best regards,
Tuuli
_______________________________________________
Geuvadis_rna_analysis mailing list
Geuvadis_rna_analysis(a)lists.crg.es <mailto:Geuvadis_rna_analysis@lists.crg.es>
http://davinci.crg.es/mailman/listinfo/geuvadis_rna_analysis
_______________________________________________
Geuvadis_rnaseq mailing list
Geuvadis_rnaseq(a)lists.crg.es <mailto:Geuvadis_rnaseq@lists.crg.es>
http://davinci.crg.es/mailman/listinfo/geuvadis_rnaseq
_______________________________________________
Geuvadis_rnaseq mailing list
Geuvadis_rnaseq(a)lists.crg.es <mailto:Geuvadis_rnaseq@lists.crg.es>
http://davinci.crg.es/mailman/listinfo/geuvadis_rnaseq
--
Dr. Michael Sammeth, Functional Bioinformatics
Phone: +34-934-020-580, http://www.cnag.eu
Centre Nacional d'Anàlisi Genòmica (CNAG), PCB
Baldiri Reixac 4-6, Room 02A8, 08028 Barcelona
Dear all,
As a reminder, we have a Geuvadis RNAseq TC today (14th) at 4 PM.
On the agenda:
- update on sequencing progress
- miRNA quality
- RNAseq meetings
April 16-17 in Geneva, future meetings?
- cell line & RNA extraction costs
- abstract for Biology of Genomes meeting at Cold Spring Harbor
- collaborations
best regards,
Tuuli
--
Tuuli Lappalainen, PhD
Department of Genetic Medicine and Development
University of Geneva Medical School
CMU / Rue Michel-Servet 1
1211 Geneva 4
Switzerland
Tel. +41-(0)22-3795550
tuuli.lappalainen(a)unige.ch
Dear all,
please find attached a very basic QC for the 5 common samples plus an
extra one (NA20524). We have not been able to fully test the pipeline in
order to deliver this now, but it should be fine.
We have seen different length profiles on the samples we have sequenced
and 4 of the common ones have a more or less similar distribution. You
will see that the extra one has very few reads and a different profile
(mode at 15bp, also seen in NA20527).
Workflow:
1) Trimming -> using far (fast adaptor removal) with a minimum of 6
bases overlap and a maximum mismatch of 2 bases every 10. We have done a
very harsh trimming for this test; we can probably make it more strict
and with several rounds.
2) Selection of uniquely mapping reads (mapping done with GEM+Bfast).
3) Generation of mature miRNA coordinates with tool from
http://cm.jefferson.edu/downloadables/
4) Reciprocal comparison (unique reads VS mature miRNA) with bedtools
(only miRNA counts are attached here)
The sequence that can clearly be read
(CGCGACCTCAGATCAGACGTGGCGACCCGCTGA) has a "perfect" match with megablast
on human genome.
Best wishes,
Sergi
---------------------------------
Sergi Beltran Agulló
Bioinformatics Analysis Group
CNAG - National Center for Genomic Analysis
Parc Científic Barcelona - Torre I
Baldiri i Reixac 4, 2a p.
08028 Barcelona
(+34)934033748
sbeltrana(a)pcb.ub.cat
www.cnag.cat
---------------------------------