Illumina Sequencing

Next generation 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 2000 (high content)
2x Illumina HiSeq 2500 (high speed)
1x Illumina MiSeq (long read)
1x Illumina NextSeq 500

Sample Clustering:

4x Illumina CBot

Fragmentation:

Covaris S2, Hydroshear

Lab on Chip:

Agilent BioAnalyzer, AAT Fragment Analyser

Fluorometer:

Invitrogen QuBit

 

Detailed sequencer description
Sequencer Lanes Regime* Read Length [bases] Reads/Lane Run time**
HiSeq 2000 8 SE, PE 50, 100 150 - 200 Mio 50 SE: 3 days
          100 PE: 13 days
HiSeq 2500 2 SE, PE 50,100 100 - 120 Mio 50 SE: 1 day
          100 PE: 4 days
           
HiSeq 2500 rapid run mode 2 PE 250 > 300 Mio 250 PE: 3 days
           
MiSeq 1 PE 36,75,150,250 15 - 20 Mio 36 PE: 8 hours
           
NextSeq 500 HI 1 PE HI: 75, 150, 300   75 PE:
          150 PE:
          300 PE:
NextSeq 500 MID 1 PE MID: 75,150,300   75 PE:
          150 PE:
          300 PE:

*) SE = Single End, PE = Paired End
**) without data processing, clustering and library preparation

 

 

GeneCore can prepare sequencing libraries for the following applications:

GeneCore can only partially support its users with processing of samples for these applications:

Target Capture

Multiplexing / Barcoding

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)

Points to consider:

 

Applicationspecific Recomendations
Application Recommended Reading Length and Mode
gDNA-Seq (WGS) 100 PE, regardless if genome assembly is based on a reference or carried out de novo
Exome Sequencing (WES) 100 PE
Methylome Sequencing (BS-Seq) 100 PE
ChipSeq, Faire-Seq, DNaseI-Seq, MN-Seq, ... 50 SE, pair-end sequencing or longer reads normally do not improve results. The sequencing depth required, is lower for analysis of binding sites of a transcription factor (not such a frequent event), whereas it is higher for mapping histones modifications ( a very frequent event).
RNA-Seq, mRNA-Seq 50 SE, for detection of expressed genes at eukaryotic samples
RNA-Seq, Splice Variant detection 50 PE and deeper coverage
miRNA-Seq 50 SE, Confirm, if your samples contain full complement of short RNAs (columns!)

 

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. Only samples which have been regeistered electronicaly can be processed. In house user may place their sample with us at V106. Please attach a corresponding barcode label available from the barcode printer in GeneCore. External users should send their sample clearly marked with the ID assigned to their sample during its registration (this information is also available in  email confirmation they receive upon completion of registration), 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). Everything else causes substantial additional work and costs for all of us.



Quality of the source material for library generation by GeneCore:

RNA-Seq

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

Chip-Seq

Purified IP DNA with majority of fragments within size range 200 - 500 bp

gDNA-Seq

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

methyl-Seq

Majority of fragments within size range 200 - 400 bp

User made libraries (all types)

BioAnalyzerRun

 

 

Required starting amount of nucleic acids
Type of Library Amount Concentration [ng/µl]
RNA-Seq (mRNA, strand-specific RNA) 1 [µg] 100
miRNA-Seq 1 [µg] 100
Chip-Seq 5 [ng] 0.3
gDNA-Seq 1 [µg] 100
Mate-pair DNA libraries 5 [µg] 100
Methyl-Seq (BS-Seq) 5 [µg] 100
Exome Capture 3 [µg] 100
PCR amplicons 20 [ng] ---
User made/ready-to-run libraries 20 ng (10 nM solution) 2

Important Points to consider during registration

What can you expect ?

Number of reads
One HiSeq 2000 flow-cell can sequence seven ‘samples’ in individual lanes and requires a control in a separate lane. These ‘samples’ can be also pools of barcoded samples. Under optimal conditions (cluster density, etc.) it is feasible to obtain from this instrument up to 200 million sequencing reads per lane. Depending upon the application, usually ~75% can be mapped to the reference genome. Please take into account that these numbers are after quality filtering.
Output
Result package contains sequences in a fastaq format and if the alignment option was selected during registration also alignments (bam file, http://samtools.sourceforge.net/) 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 necessary to generate the result files. The result package will be available 30 days after you receive the message informing you that data are ready. A longer storage is not possible due to the amount of data produced and the respective costs for data storage. Data are archived and can be retrieved against the fee, if a need be. Please note that generally we do not manipulate data by any means such as trimming, for example. However, we can do it upon request.
Turnaround time
is heavily dependent upon the sequencing regime. Running time of the sequencer for 50 single-end bases (50SE) is ~3 days, for 100 pair-ended bases (100PE, each fragment is sequenced from both sides) 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.

Result download
The files are available as fastq file *.txt.gz and bam files and the names have the following notation:
FileNameNotation

Results are placed on our dedicated result distribution server. The files can be downloaded, using with the fasp protocol (by Aspera), which increases the donwload speed up to a factor of 60x (compared to conventional methods), utilizes enhanced error correction as well on the fly encryption. For further information: information File Download with Aspera.pdf.

Sample storage policy
Your physical sample and generated sequencing library will be stored at GeneCore for a period of 30 days after completion of your request, if no explicit agreement with GeneCore has been made.

Data retention policy
As soon as the result files for your samples are produced (seq and if applicable also bam filess) you will get a notification mail. We will keep your data online for download for 4 weeks, after that period the data files will be deleted.

Pricing
MPS on Illumina HiseSeq consists of two parts: library preparation and sequencing. . Each of them has its own price, which depends upon the type of the library and its sequencing mode.