February 3, 2018 - New QC criteria: rRNA (28S/18S) NEW

To improve the Initial QC of RNA samples, a new QC criteria has been implemented. To predict library preparation success, we are going to  use the 28S/18S ratio (in addition to the RIN) in the future. RNA samples with a 28S/18S ratio lower than 1.0 will fail the initial QC. Using the 28S/18S ratio in addition to the RIN can reduce the number of false positives (number of samples that have been incorrectly predicted to yield a library (RIN >= 8)) by about 10%.

This cut-off only applies to total RNA submissions (not depleted RNA, small RNA or Ultra Low RNA).

January 10, 2018 - Galaxy bioinformatics platform at the GPCF

The Genomics and Proteomics Core Facility (GPCF) can now offer users at the DKFZ and in the DKTK our local implementation of the Galaxy bioinformatics platform for analysis of Next-Generation Sequencing and other genomic data.

The system is designed to support data-intensive biomedical research and allows users to customize their own bioinformatics workflows using a graphical pipeline builder. We will support our users in all aspects of data analysis ranging from the import of your sequencing data to choosing the tools most appropiate for your research. The system and its graphical interface will officially be introduced at an upcoming GPCF Tech-Talk.

Should you be interested in using the GPCF Galaxy instance we would ask you to contact us at: htseqbioinf@dkfz-heidelberg.de.

November 9, 2017 - New WGBS Protocol

Starting in January 2018 we will replace our standard bisulfite (WGBS) protocol with the new Accel-NGS® Methyl-Seq DNA Library Kit from Swift Biosciences. With this kit, only 500ng of DNA input material will be required. This is 10-times less than what is needed for our current protocol. In addition the average coverage will be higher due to a larger library size. We thoroughly compared sequencing results of the old and the new protocol, and found no significant difference. If you have samples which still need to be prepared with the old protocol please get in touch with us (a.schulz@dkfz.de).

October 24, 2017 - Strand specific depleted RNA/Poly A RNA

We now offer a protocol for strand specific sequencing of depleted and Poly A enriched RNA. This is a modified version of the Illumina TruSeq mRNA strand specific protocol. We need 20ng of depleted RNA/Poly A RNA as input material. Please note that even though we will perform an initial QC we can only evaluate if the rRNA depletion/Poly A enrichment was successful. The RIN can only be calculated from total RNA. We therefore recommend that customers perform a QC measurement with Bioanalyzer or Tapestation, prior to depletion or Poly A enrichment. The protocol will replace the non-stranded depleted RNA protocol. Please note that the first 200 samples are treated as R&D.

September 19, 2017 - Christmas 2017 closure

Dear customers, as every year we need to shut down our facility for some maintenance work.

The sequencers will be shut down on Monday December 18th. We will successively wake them up again after New Year’s. We expect to have regular workflows from January 8th on again.

For Fastrack submissions the last regular sample delivery day will be December 6th, the first regular delivery day in 2018 will be on January 10th. Samples submitted in between will have longer than usual turnaround times.

Our QC will be closed from December 21st until January 1st.

Please get in touch with Angela Schulz (a.schulz@dkfz.de) if you have any questions.

August 30, 2017 - Dual Indices for DNA and RNA-Seq

Due to the adapter hopping issue occuring on patterned flowcells (HiSeq X and HiSeq 4000), we have now switched to unique dual index (UDI) adapters for the prep of DNA libraries (TruSeq Nano DNA) and RNA libraries (TruSeq stranded mRNA HT).

This allows us to drastically reduce the effects of index hopping bioinformatically, as only reads with matching adapters on both ends are considered. Please also see our FAQs about Adapter-Hopping.

At the moment there are 8 unique dual indices available. This means that there are 8 combinations where both i7 and i5 have unique sequences. Therefore, up to 8 RNAs can be pooled. Please see our Pooling Recommendations for more details. This allows for example pooling of RNAs on HiSeq 4000.

We also offer 50SR runs on this platform (610 Euro/lane).

June 22, 2017 - Data download server address change

The download link and the adress of the midterm download server, where you may access your sequencing result data, has been changed:
The prefix "pool0." needs to be replaced with "midterm.".

The old address was: \\pool0.fast-isilon1.dkfz-heidelberg.de\W190-MidTerm\6_DIGIT_SUBMISSION_ID

The new address is: \\midterm.fast-isilon1.dkfz-heidelberg.de\W190-MidTerm\6_DIGIT_SUBMISSION_ID

For more details, read the updated FAQ entry: Data Access - How can I access / download my sequencing result data?

May 11, 2017 - MiSeq 300 PE V3 discontinued

As we observed massive problems concerning stability with the 300 PE kit, we will remove it from our portfolio until Illumina resolved the problem.

April 20, 2017 - HiSeq X / 4000 Multiplex - Adapter Hopping

Pooling on patterned flowcells (HiSeq X and HiSeq 4000) may lead to misassigned reads:

Recently a paper by Sinha et al [1] came out describing a problem that free adapters cause wrong assignment of reads on sequencing platforms using patterned flowcells (HiSeq X; HiSeq 4000). Those lead to contaminations in between multiplexed samples. The more free indices are present in the samples/lane, the more contaminations occur.

In our FAQ page, you can find more information about this problem, what we are doing to detect and prevent it, and recommendations what you should do when submitting multiplexes for HiSeq X or 4000.

[1] Sinha et al: Index switching causes “spreading-of-signal” among multiplexed samples in Illumina HiSeq 4000 DNA sequencing - https://doi.org/10.1101/125724

April 10, 2017 - New Software on HiSeq X

Illumina has released a new software version for HiSeq X (HCS 3.4.0) to overcome the problems with bisulfite libraries. Now only 5% PhiX spike in is necessary, which results in average coverages of 18 fold and more per lane. For all other applications on the HiSeq X we continue to spike in 1% PhiX. We validated that data created with different software versions are comparable.

February 06, 2017 - New standard RNA-Seq kit

Instead of the Illumina TruSeq kit we used so far we will switch to Illumina’s mRNA TruSeq stranded kit from mid of February on. We thoroughly compared both kits and didn’t find significant differences in expression profile, duplication rate, and alignment rate of the samples, but you get the extra information about strandness. Also, the kit is more stable with lower input amounts.

To control for strandness ERCC spike in (synthetic RNA) will be added (Synthetic spike-in standards for RNA-seq experiments). If you submit samples for RNA Seq this protocol will be used. The Agilent stranded kit will be discontinued.

November 17, 2016 - New Software Update on HiSeqX

Illumina updated the software on the HiSeqX machines. The new software requires a PhiX spike-in of 30% for bisulfite sequencing instead of the 10% that were necessary with older software versions. Therefore, the read amount per sample and the coverage that can be achieved on one lane is approximately 30% reduced. For whole-genome-sequencing the amount of PhiX that needs to be spiked in remains the same as before.

April 1, 2016 - HiSeq 4000 Paired-End 100bp

We now offer sequencing on our new Illumina HiSeq 4000 machines!

Please read the FAQ entry HiSeq 4000 - Applications for important information concerning this type of sequencer.

March 30, 2016 - Low Input Exome-Seq Human V6 ± UTRs

Two new exome-seq application types (announced on February 8) are now available:

  • Low Input Exome-Seq Human v6
  • Low Input Exome-Seq Human v6+UTRs

February 17, 2016 - Prices 2016

Our financial department has calculated new prices for library preparation and sequencing and we are happy to announce that the prices for in-house customers dropped for all applications. Only material costs will be charged. Furthermore, there are no longer different price categories (DKFZ-A, DKFZ-B, DKFZ-C, DKTK-A and DKTK-B) for basic and third party funded projects. Of the former seven different types of funding only three are left and most of the time FT1: DKFZ or DKTK will be the right choice for you.

All prices are retroactively effective from January 1st 2016!

February 16, 2016 - Delays for RNA-Seq and all HiSeq 2000 V3 sequencing types

Since Illumina has severe supply difficulties with the RNA TruSeq Kit, we cannot prep any RNA-Seq libraries at the moment. Unfortunately we have no further information from Illumina about when the problem will be solved. We will finish your samples on a first come first serve basis as soon as the new kits arrive. The protocols for small RNA, strand specific RNA and Ultra low RNA are not affected by Illumina's supply difficulties.

Since the beginning of 2016 we only have one HiSeq 2000 V3 machine left, so the turnaround times for this sequencing type are longer than usual. One paired-end sequencing run on this machine takes about 2 weeks and we can only sequence 16 lanes per run, which leads to delays in data delivery.

February 8, 2016 - Exome-Sequencing News

There are two important changes concerning Exome-Sequencing in our Core Facility Unit:

Exome V4 discontinued
Since beginning of 2016 we have discontinued Agilent SureSelect V4 for exome enrichment. We still offer a limited amount of V4+UTR and both V5 versions. In the near future V6 and V6+UTR will be added to our portfolio as well. The new version has several advantages like optimized bait selection for more uniform coverage and an updated content, including challenging regions. In case you have started a project with V4 it is not an issue to switch to V5 or V6.

Switch to Low Input Exome-Seq protocols
In order to speed up our turnaround times we will only offer the low input protocols (V5 and V6 with and without UTR) for exome enrichment from March 1st on. We thoroughly compared both protocols (normal and low input) and couldn’t find significant differences in the results. The low input protocols have the advantage that they only require 450 ng of DNA instead of 3000 ng.

December 15, 2015 - Switch from Tubes to Plates

In order to coordinate the increasing number of samples as well as to further improve our accuracy and efficiency we will switch to 96-well plate format for all incoming samples and libraries (except multiplexes). For a smooth transition please keep in mind the following points:

  • From January 1st 2016: deliver your samples and libraries in 96-well plate format (use tubes for multiplexes as before)
  • Exclusively accepted plates are: Eppendorf twin.tec® microbiology PCR Plate 96, skirted, 150 µL, clear (Order no.: 0030129300)
  • Make sure the plates are completely closed with the accompanying seal ("Eppendorf PCR Film (selbstklebend), 100 pieces", Order no.: 0030 127.781) and use spatula
  • Place samples into plate according to the received plate layout (column by column; depends on sample order in submission)
  • Until the end of 2015: continue to deliver your samples in 1,5ml safe lock low-bind Eppendorf tubes

February 8, 2016 Update:
We had to switch to another kind of "Klebefolie" for the plate seals: ["Klebefolie hitzefest zum Versiegeln von PCR Platten"; Order no.: SL-AM0558] had to be replaced by ["Eppendorf PCR Film (selbstklebend), 100 pieces" (Order no.: 0030 127.781)].

June 8, 2016 Update:
There is a new and better kind of seal available now: "4titude PCR Foil Seal Strong", Order no.: 4ti-0500FL.

December 2, 2015 - Holiday notice on Christmas

Due to required maintenance work we will have to shut down the facility and therefore will not be able to accept or receive any samples/libraries/multiplexes between December 24 and January 10. New submissions will be possible.

December 1, 2015 - HiSeq 4000 available soon!

We are happy to announce that we will have a new Illumina HiSeq 4000 available as of the beginning of 2016. This machine has several advantages compared to the currently used HiSeq 2000 V4 machines. The run time of the HiSeq 4000 is about 3.5 days for a 150 paired-end run compared to the HiSeq 2000 V4 which needs about 6 days for a 125 paired-end run. Meanwhile you can get a maximal output of 700M reads compared to 500M reads on the HiSeq 2000 V4. This means you can e.g. pool up to 7 human exomes (without UTR) per lane with an estimated coverage of 80x. For details about specifications please check our price list . The price per lane of the HiSeq 4000 will be very similar to the one for the HiSeq 2000 V4, so the price per base will noticeably decrease. The HiSeq 4000 platform will be open for the following applications:

  • Whole Exome sequencing (all versions)
  • RNA sequencing / strand-specific RNA sequencing
  • Ultra low RNA sequencing
  • Small RNA sequencing
  • ChIP sequencing
  • Library + Multiplex sequencing of the above mentioned applications
We will make 150 paired end available as a sequencing type as soon as the new HiSeq 4000 sequencer has been installed. The HiSeq 2000 v4 will still be available with 125 paired-end and 50 single-read runs. For whole genome sequencing and whole genome bisulfite sequencing we recommend to use the HiSeq X platform (open for all species).

October 9, 2015 - Discontinuation Information

The following two sequencing types are as of now obsolete and can no longer be provided:

  • HiSeq 2000 v3 Single-Read 100bp
  • HiSeq 2000 v3 Paired-End 50bp
Kindly excuse any inconvenience.

August 15, 2015 - New "midterm" storage server

After your submission has been completed, your data will be readily available at the midterm storage server for at least 3 months.
We will assist you with moving your data to the long-term storage provided by the ITCF where you have read and write access to analyze your data.

Please read the new FAQ entry for more information: Data Access - How can I access / download my sequencing result data?

August 15, 2015 - Data access rights

The submitter and project coordinators can now modify the data access rights of their submission.
Please read the new FAQ entry for more information: How can I give other users access to my sequencing result data?

July 01, 2015 - Sequencing Price Reductions

Since the intensive negotiations between the DKFZ and Illumina have payed off, we are happy to announce, that as of now the prices for sequencing can be lowered by 6% (on average). The sequencing type HiSeq 2000 v4 Paired-End 125bp is now 100 € cheaper than before for example. All the new prices are available via our usual Price List and are effective immediately for all new submissions.

June 18, 2015 - Announcing end of SureSelectXT Human All Exon V4 and introducing V6

Agilent has announced, that they will discontinue the Exome Library Preparation Kit "SureSelectXT Human All Exon V4" in 2016.

These application types will not be available anymore in 2016:

  • Exome-Seq (SureSelectXT Human v4)
  • Exome-Seq (SureSelectXT Human v4+UTRs)
  • Low Input Exome-Seq Human v4
  • Low Input Exome-Seq Human v4+UTRs

At the same time, we want to introduce the new version V6 of this kit for Exome Library Preparation: SureSelectXT Human All Exon V6
Also, version V5 will stay available in 2016. We strongly recommend to use V5 for new projects.

Please contact us, if you still need the old version V4.

April 01, 2015 - New Sequencing Prices and Types

Illumina has updated its price list for core consumables. Since the new prices are considerable higher than before we had to update the prices for our HiSeq Lanes and MiSeq Runs as well. The new prices are available via our usual price list and are effective immediately for all new submissions.

As of now there are also two new sequencing types available:

  • HiSeq 2000 v4 Single-Read 50bp
  • HiSeq 2000 v4 Paired-End 125bp
Both new sequencing types make use of Illuminas HiSeq v4 kits which allow longer reads, approx. 20% more reads and 40% faster sequencing runs compared to HiSeq v3 kits. Please be aware that our old HiSeq v3 sequencing types, including the frequently ordered sequencing type HiSeq 2000 v3 Paired-End 100bp, will only be available till the end of 2015!

January 28, 2015 - New FAQ entries on Library QC and Multiplexing

There are two new entries available on our FAQ page. The first about which devices, procedures and material to use when doing a Libary QC and the second about multiplexing. The latter also contains a link to an excel sheet which might be of use to you and your calculations, if you are not entirely sure how to normalize your libraries to a certain molar concentration before the actual multiplexing step.

Here are the direct links to the new entries (need login):

January 12, 2015 - New Sample Requirements

As announced in our October newsletter we have updated our volume, concentration and mass requirements for unpreped samples. Please refer to our price list for the new base material and application type specific values before you submit any new samples.