Agreement
for Illumina
NGS RNA-seq/Small-seq collaboration
UMR INRA 116 -UEVE-ERLCNRS 8196
Validated by S. Balzergue :XX.XX.XX
General information and conditions of access
Access to mRNA-seq sequencing for transcriptome analysis is possible through partnerships
between the URGV and other laboratories, within the extent of the resources produced in URGV.
To benefit from this support, interested teams will fill a request form to be sent to Sandrine
Balzergue (URGV) by email (balzerg@evry.inra.fr).
The preparation of libraries, sequencing on Illumina units will be performed by dedicated staff of
the platform. The team brings in its expertise on transcriptome analysis and its workforce in this
collaboration but does not benefit from sufficient financial resources to support the cost of
consumables that will therefore be charged to the partner laboratory:
RNA-seq protocols :
100€ per library « classical » TRUSeq Illumina
110€ per library « stranted specific » TRUseq Illumina
150€/library « Small Illumina »
55€ /library “NEBNExt Small New England Biolabs”
(Please, Contact the Platform if specific protocol is needed)
Sequençing :
1500€ per lane of HiSeq2000 Pair End 2x100b (+/- 150000K reads)
1900€ per lane of Hiseq2500 Pair End 2x150b (+/- 150000K reads)
1000€ per lane of Hiseq2500 Single Reads 1x100b
Bioinformatics and statistical analysis :
Option B1
100€/sample for bioinformatic analysis (mapping on transcriptome), differential analysis (2 by 2)
and, maintenance costs of server and data storage.
Option B2
200€/sample for bioinformatic analysis (assembling + mapping on contigs or mapping on genome),
differential analysis (2 by 2) and, maintenance costs of server and data storage..
For this 2 options, raw data are storage and kept at URGV throughout the project + 1 year after
sending the results to collaborators
Option B3
100€/sample for bioinformatic analysis on Small RNA (mapping on reference genome),
differential analysis (2 by 2) on known miRNA and, maintenance costs of server and data storage.
Option B4
200€/project : for collaborator who haven’t ask for bioinfo/statitic analysis, it is possible to have a
storage of the raw data at URGV during 6 months (limited to 1Terra) .
Note: A quote can be made and sent to collaborator only if requested to Sandrine Balzergue
(balzerg@evry.inra.fr).
The collaborator must edit a purchase order in the amount specified by this quote and in a delay of
15 days after receipt of samples. Collaborator must send the original order at INRA-URGV,
accounting department, 2 rue Gaston Cremieux 91000 Evry. If you want more information, please
contact: secretariat@evry.inra.fr
Version du 10.12.2013-S. Balzergue
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Agreement
for Illumina
NGS RNA-seq/Small-seq collaboration
UMR INRA 116 -UEVE-ERLCNRS 8196
Validated by S. Balzergue :XX.XX.XX
Data exchange format:
The raw data, contigs (if performed), raw counts, functional annotation of UniGene set (if requested
option) will be available via our secure FTP site loading.
The collaborator agrees to repatriate the raw data on their own server maximum one month after the
date of data availability.
Normalized data and the results of the differential analysis will be sent as an Excel table via FileX
with a password.
Important: For collaborator who have not requested bioinfo / stats analyzes : the data will not
be backed up URGV except if B3 option was taken. Upon request, the URGV platform can
make a backup of the raw data (backup of FASTQ) for a time of 1 year (cost: 200 € / project).
Data storage:
The URGV agrees to keep the raw data (not images) for 1 year after completion of the project.
After this period, data will be deleted.
Databases:
It is expected that the results of experiments are integrated into the database CATdb, Gagnot et al.
Nucleic Acids Res. January 2008, 36 (compatible with standard MIAME: Brazma et al, 2001. Nat
Genet. 29 (4) :365-71) and transmitted to the Geomnibus (GEO) database of NCBI. GEO will
then issue an accession number required for any publication of transcriptome results. Be careful if
you do not plan to publish all data at the same time, fill 2 different files for two accession numbers
(if needed contact us for more information.).
Data Release :
The data will be released within 1 year after project completion; an email will notify you 15 days
before. On the same date, the remaining RNA samples and libraries will be returned.
Nevertheless, there are exceptions that will be discussed before the beginning of each project:
1) if it’s an industrial collaboration
2) if it is within the framework of an ANR / KBBE project and the results are placed in the public
one year after the end of the project itself only.
3) if the transcriptomic results has a value for filing patent
Technical data (protocols etc..) and a list of current collaborations are available on the URGV
website: http://www.versailles.inra.fr/urgv/microarray.htm
Characteristics of sequencing runs and delays
The runs are performed on the Illumina sequencer HiSeq2000 or Hiseq2500 via the CNS Genomics
Institute in Evry.
The number of reads per sample is adjusted according to your biological question.
A phone call or a meeting at Evry to determine the aims and issues of the project and set up the
experimental plan will systematically take place. This meeting should include the collaborator, the
transcriptomics platform member and bioinformatics team of URGV.
A second tripartite meeting will be scheduled upon receipt of sequences.
Schedule:
The estimated time from reception of RNA of satisfactory quality is about three months to construct
libraries and sequencing data. Depending on the options chosen for Bioinformatics analyzes a
further period of two months is required. This time takes into account:
- - Construction of librarie
- Sequencing
Version du 10.12.2013-S. Balzergue
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Agreement
for Illumina
NGS RNA-seq/Small-seq collaboration
UMR INRA 116 -UEVE-ERLCNRS 8196
Validated by S. Balzergue :XX.XX.XX
- - Analyses bioinformatics following option selected
- Statistical analysis: normalization and differential analysis of data
- Data Integration into CATdb and GEO
Samples preparation
It is important to note that many factors influence the gene expression levels of a plant. The control
of experimental conditions is therefore crucial if we want to link a difference in expression to the
function studied. And a control plant compared to a plant having undergone a specific treatment
should be grown in the same light and nutritional environment as the latter. For example, a shift of
sampling time during the day will reveal differences due to the circadian expression of many genes.
A lack of homogeneity of watering or plant treatment can be a source of variability unrelated to the
studied process. These considerations should be taken into account to ensure the reproducibility of
sampling.
Replicates
It is essential to distinguish between a technical repetition and biological repetition.
Technical replicates:
Technical repetitions of a sample are prepared at the same time (sowing, harvest, extraction ...).
Samples from different individuals but from the same experiment are considered as technical
replicates
It allows the observation and quantification of technical bias (technical variability), the control of
the reproducibility of the study and the quality control of the data but the results cannot be
generalized.
We also carry out the sequencing of biological replicates on different tracks.
Biological replicates:
Biological replicates samples are prepared in independent experiments (sowing, harvesting,
extraction ...) with at least a 24 hours shift (beware of the circadian cycle).
It allows the observation of inter-individual and inter-experiment variability.
The results can then be generalized.
It is necessary to provide at least one biological repetition, that is to say a repetition of the whole
experiment. The aim is to characterize the biological variability between replicates, and "remove" it,
to identify genes whose differential expression is related only to the studied factor.
Quantity and quality needed for experiments
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Agreement
for Illumina
NGS RNA-seq/Small-seq collaboration
UMR INRA 116 -UEVE-ERLCNRS 8196
Validated by S. Balzergue :XX.XX.XX
4μg of total RNA (minimum concentration of 200ng/μl, contact the platform if you are not able to
get this amount) per sample is expected. The purity of RNA is one of the most important factors for
the success of the experiment, it is preferable to use an affinity purification protocol including the
step of DNase I treatment (RNeasy kit Qiagen)
For the sequencing of small RNA => We recommend to performed extractions with
MIRVANA ™ miRNA Isolation Kit (Ambion), an amount of 2μg at 170ng/μl minium is
required).
For "difficult" samples such as seeds, the addition of PVP is very useful, contact us if needed. Total
RNAs will be sent in dry ice in the elution solution. Their quality will be estimated on Agilent Chip
and will be quantified with "RiboGreen" after arriving on the platform.
Total RNA or Small RNA will be sent together with the fully completed information table (see
and print the last page of this agreement).
Process
- Quality control of total RNA (Agilent Bioanalyzer) and quantification (RiboGreen).
- Construction of libraries (RNA-seq, Small-RNA, RNA-seq-directional ...): Illumina protocols
mainly
- libraries Quality checking on Qbit et Agilent Bioanalyzer
- Preparation of samples for sequencing (c-bot)
- Sequencing on Illumina HiSeq2000 or Hiseq2500
- Assembly(if needed)
- Contigs (if needed)
- Mapping
- Counting
- Standardization with TMM
- Differential Analysis (EdgeR and HTSDiff)
After statistical analysis of raw results, for each comparison, a list of genes is produced as an Excel
file. It includes the average count of condition #1, the average count of condition #2, the ratio, a raw
and adjusted p-value to allow control of false positives.
Results publication
This is a project involving the collaborating scientists and the URGV, in which the platform brings
in its expertise. Only the cost of consumables is supported by the partner laboratory.
- A member of the transcriptomics Platform and a member of "Genomics and Predictive
Bioinformatics" of URGV will be co-authors of the first publication in which transcriptome data
will be presented. The same convention will be applied to the filing of Patent on the initiative of
the employee and in which the transcriptome results will be used.
You will also be asked to include in the description of the data, the CATdb database (example: "All
raw and normalized data are available-through the database CATdb (AU_XXXXXXX, Gagnot et
al, 2008) and from the Gene Expression omnibus (GEO) repository at the National Center for
Biotechnology Information (NCBI) (T. Barrett et al. NAR 2006): GSE accession number XXXXX.
Version du 10.12.2013-S. Balzergue
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Agreement
for Illumina
NGS RNA-seq/Small-seq collaboration
UMR INRA 116 -UEVE-ERLCNRS 8196
Validated by S. Balzergue :XX.XX.XX
Project Design (required):
We ask you to provide the following information:
1 - Title of Project
2 - Name and address of project manager
3 - Name and address of the person responsible for monitoring the analysis in relationship
with URGV
4 - Scientific aims (be as accurate as possible including :)
Biological question?
Annotation, RNA quantification / Small-RNA, construction of High Density chip? ….
5 – Experiment Design including:
Number of reads per sample:
Type of library:
Sequencing machine:
Multiplexed sample (how?):
Sequencing: Single Reads / Pair End
Sequencing length: 100bp, 150bp
Does a reference genome or UniGene (transcriptome) set exist?: yes / no
…
6 - Number of libraries - description of samples per run (organ, stage of sampling according to
Boyes et al. Plant Cell 2001, treatment ...)–
7 - Expected date of delivery of samples to URGV
Signature:
Experimental laboratory(URGV)
Collaborator
-----------------------------------------------------------------------------------------------------------------------8 – Table to join to the RNA samples when sending:
TUBE name
Sample
name
experimental design
Version du 10.12.2013-S. Balzergue
on Concentration µg/µl
RNA
extraction
method? DNAseI?
Page 5 / 5