Solexa

High throughput sequencing has become a relevant technology in modern biology. GeneCore is providing 'Massively Parallel Sequencing' (MPS) as a core part of its service portfolio according to EMBL's mission to provide a state-of-the-art infrastructure to the scientific community.

The fast growing suite of instruments consists currently of:

Sequencer:

3x Illumina HiSeq
  1x Illumina GAIIx with paired end option

Sample Processing:

3x cBot

Fragmentation:

Covaris S2, Hydroshear

Lab on Chip:

Agilent BioAnalyzer

Fluorometer:

Invitrogen QuBit

GeneCore can prepare sequencing libraries for the following applications:

These library types have to be provided by the users:

Target Capture

Sequencing modes (read length 36 to 100 bases)

Multiplexing

User made libraries

 

Which type of sequencing to use ?

"If you don't get the coverage at the start you'll regret it." (Jonathon Blake, Bioinformation)

Sequencing Type Reccomendation

Exon Capture

40 MB Kit, human: single-end, 72 bp
50 MB Kit, human: single-end, 105 bp (to get sufficient coverage)

Whole Genome Sequencing

Large rearrangements: Mate pairs, large inserts
Resequencing: paired-end, 100 bp+
SNPs: paired-end, 100 bp+
Indels: paired-end, 100 bp+

RNA-Seq

Tag counting:
Large number of mappable tags, single-end 36-50 bp should be sufficient. Longer reads may need to be trimmed to match exons.
Transcriptome assembly or exon usage:
Single-end 75+ or paired-end 50 bp+ depending od de novo /spliced read mapping approach or a map pairs to different exon approach.

Chip-Seq

single-end, 36 bp, unless you have real concerns about alignability of your target (i.e. some strange looking enhancer region)

Multiplexing

The barcode reduces the numer of final bases (currently 6 bp). It also reduced the coverage by the multiplexing factor respectively. Useful, if you know, that you get sufficient read coverage from a lane to provide enough depth for a number of samples.

 

 

What can you expect ?

Number of reads
One GAIIx flow cell can sequence seven libraries in individual lanes and requires a control in a separate lane. Under optimal conditions (cluster density, etc.) up to 20 million sequences per lane are generated, from which usually ~70% can be mapped to the reference genome. Please take into account that these numbers are after quality filtering.
Output
Result package contains sequences and alignments as a tar archive. You will be notified by email when files are ready for download. Primary and intermediate results will be stored as long as necassary to generate the result files. The result package will be availabe 30 days. A longer storage is imposible due to the amount of data produced and the respective costs for data storage.
Turnaround time
may vary but is heavily dependent upon the running time of the sequencer and the data processing. The sequencers are running 24 hours a day and 7 days a week. Interruptions are only made for maintenance. The general processing time per sample is around 6 weeks, exceptions are possible.

Sample storage policy
Your physical sample and generated sequencing library will be stored at GeneCore for a period of 6 weeks after completion of your request and charging, if no explicit agreement with GeneCore has been made.

Data retention policy
As soon as the result files for your samples are produced (export &, seq files) you will get a notification mail. We will keep your data online for download for 6 weeks, after that period the data files will be deleted.


What do we need ?

First Time
Please contact Vladimir Benes , if you like to use GeneCore for MPS
Ordering
Please use our registration system to place your order. In house user may place their sample with us at V315. Please attach your respective barcode label available in front of our lab. External users should send their sample clearly marked with the ID they get send in their email confirmation, that we can attach the proper label for processing and storage, as soon as the samples arrive.
Sample Requirements
Container: Please use only 1.5 ml low binding tubes (e.g. Eppendorf)
Quality of the source material for library generation by GeneCore:

RNA-Seq:

High quality total RNA (BioAnalyzer RIN > 7), 260/280 ~2

Chip-Seq:

Purified IP DNA with majority of fragments within size range 200 - 400 bp

gDNA-Seq:

High quality DNA, single band on agarose gel, 260/280 ~2

methyl-Seq:

Majority of fragments within size range 200 - 400 bp

User made libraries (all types)

BioAnalyzerRun

Amount:

Type of Library

Amount of starting material

Concentration

RNA-Seq

5 ug

0.5 ug/ul

Chip-Seq

10 ng

300 pg/ul

gDNA-Seq

5 ug

0.5 ug/ul

methyl-Seq

10 ug

300 pg/ul

User made libraries

20 ug

2 ng/ul

Pricing
MPS on Illumina GAIIx consists of three parts: library preparation, clustering and sequencing. Each of them has its own cost, which depends upon the type of the library and its sequencing mode.

Links

Library Preparation

Sequence Capture

Nucleic Acid Quantification

Depletion of ribosomal RNA