Next generation Sequencing
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Illumina Next generation Sequencing (INGS)
The LGTC owns two HiSeq 2000s from Illumina. The Hiseq 2000 is currently the world leading machine for generating large amounts of sequence data. This makes the hiseq suitable for sequencing large genomes or sequencing a pool of multiple barcoded DNA samples (effectively reducing the costs per sample).

The technique used by Illumina for sequencing is called "sequencing by synthesis". More information about this technology can be found in the Illumina sequencing information guide and on the Illumina website.

  • SNP detection
  • transcriptome sequencing
  • miRNA profiling
  • ChIP-seq
  • sequence capture
  • targeted resequencing
  • de novo sequencing
  • ...

INGS at the LGTC
When you have a project for INGS it is necessary to discuss it with the LGTC. We can help you discuss the possibilities, costs, planning, and all other issues. For most of the projects it is wise to start with a pilot experiment. We will also recommend you to do the sample preparation yourself under supervision of the LGTC.

Once it has been decided how to start you will need a quotation from the LGTC. This quotation can be requested via the LGTC order system for which you will need a valid account. A quotation for INGS contains the following products: sample preparation reagents, sequencing, INGS support. We do not do data analysis on a regular basis, if you want the LGTC to do the data analysis, it has to be included in the quotation. To accept the costs you need to deliver a printed and signed version of the quotation to the LGTC and for LUMC, with a valid project code. Only then, the project is agreed. The quotation you receive from the LGTC is valid for one month, when you agree with the quote you need to deliver it within a month to agree the project. If it is later than one month, costs for the project need to be reevaluated. After agreement of the quote samples need to be delivered within three months, if it takes longer than that the costs for the project will have to be reevaluated. A project needs to be finished within the year. If it is not the case we will make an invoice for the part that has been done and make a new quotation for the part that still needs to be finished. This is necessary because prices of reagnets can change without notice or when updated by the manufacturer.

Once you are ready to deliver your samples you need to submit them in the LGTC NGS LIMS. You can log in the LGTC NGS tracking system with username lgtc and password lgtc, this LIMS is accessible only within the LUMC network. A manual for the LGTC NGS LIMS can be found here. Without a valid quotation (signed and delivered at the LGTC and with a project code for LUMC projects) you will not be able to enter your samples in the LIMS and the project will not start. The project starts when your samples are submitted in the LIMS and agreed by the LGTC. You will be notified by mail. Only then you can deliver your samples at the LGTC. When you do the sample preparation yourself, the protocol you are using has to be approved by Illumina or by the LGTC. Wrong sample preps will give bad sequencing results. Once you are finished with the samples you will need to do a lab-on-a-chip and send the files with the measured molarity. Check our recommendations for good sample preps. You need to be absolutely sure of the molarity of your samples; wrong concentrations will give problems in the clustering and give bad sequencing results.

Planning and time span
According to our terms & conditions, results will be delivered within three months after the start of the project. For exceptional projects we may need more time. If that is the case for your project it will be mentioned in your quotation. The project starts when the samples are delivered to the LGTC together with the signed quotation and the sample sheet. When unexpected circumstances occur that delays the planning, you will be notified.

Data and data analysis
The quotation does not automatically include data analysis since the LGTC will not do the data analysis of your samples. If you want complete data analysis to be done it has to be included in your quotation. On the internet and via courses you can get all the information you need for INGS data analysis. We will only do the basic QC, base calling, and initial alignment of the data. From that point you will receive the data from us. For now you will be able to download it via a FTP server. Soon you will get it handed over on a DVD. The files you receive are FASTQ and SRF files with the sequencing data and quality values of the called bases. With these files you can do the data analysis, for more information see NGS Bioinformatics.

Summary: what can you expect from us? General rules
  • The LGTC will do the clustering and the sequencing. When the run has failed due to technical (machine) problems with the GAII/HiSeq2000 or the cluster station we will discuss the possibilities of repeating the run. All other causes of failed runs will not be refunded and not re-run for free.
  • You will receive FASTQ files (includes sequences and quality values of the base calling). We will only keep your data for three months, it is your own responsibility to make back up copies of the data you receive from us. All other wishes concerning the data will be considered as data analysis for which you will receive a separate quotation.
  • All discounts, special wishes, planning, complaints, problems, etc... have to be discussed with and agreed by the lab-manager or they will not be considered valid.

More information? Questions?
Go to
Contact the LGTC.

This is the full overview of HiSeq2000 problems the LGTC has encountered in the last years.
We apologize for the the delays in the projects and the inconvenience caused. We rely on illumina to solve the issues.

Friday, May18
Starting a new run on the HiSeq the solenoids broke. Repair planned for Monday 21. New run start delayed till Tuesday May 22.

May 4th
The HiSeq controller stopped scanning for over 14 hrs. Luckily this did not happen during the weekend. We could save the run but this delays the current run and all planned runs. We received no explanation from illumina why this can happen.

May 3rd
1 Flowcell done at ErasmusMC.

End of April
The air bubble problem on the second HiSeq has finally been solved. After 4 months we can now finally use this second HiSeq, now that it meets LGTC standards.

April 18th
The Solenoids broke during the run, around cycli 180.

April 5th
PC communication problems, run stopped. Techsupport advised to restart the run, that seemed to work out.

2 Flowcells run in France.

Camera time out errors. It delays the current runs and the whole planning. The problem was fixed within a few days.

February to May
All runs on the second HiSeq2000 contain disturbing air bubbles causing a significant increase in "N"s in a read.

February 17th & 23rd 2012
Validation runs on second HiSeq2000.

January 2012
The LGTC received a second HiSeq2000 from illumina to solve the issues with the first loan machine.

February 10th 2012
Low G intensity and FWHM out of spec.
December 16th 2011
Blockage of lane 4 due to fluidics issues. Solenoids (pumps) replaced. Run stared with a delay due to the repair of the solenoids.
November 16th 2011
Blockage of fluidics (solenoids), sudden drop of q values at start of read 2.

November 15th 2011
Fluidic (solenoids) problems in 2 lanes.

November 1st 2011
Fluidic (solenoids) problems in 3 lanes.

October 5th 2011
RTA slowed down. Delay in the current run.

October 4th 2011
Read2 quality was bad, tried to reset the run but Read 2 failed.

September 30th 2011
Problems with cBot, clustering restart.

August 25th 2011
Problem with single end flowcell, fluidics problems in some lanes.

July 18th 2011
Validation runs on second HiSeq2000.

July 14th 2011
The LGTC received a second HiSeq2000 from illumina to solve the issues with our own machine.

June 9th 2011
The HiSeq2000 is broken again: the laser will be replaced. This will take a week to get fixed.

May 30th 2011
The HiSeq2000 at the LGTC has problems on a regular basis. We are processing samples to get rid of the waiting list but runs are not always successful.

Feb 2011
Due to complications with the HiSeq2000 we are encountering unexpected delays to process the samples.

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