News

Date: 11. December 2017

Subject: Limited sample receiving capacities during Christmas time

Dear NGS user,

We wish a merry Christmas time to all our customers and collaborators!
Please note: From 20.12.2017 until 02.01.2018 no new samples or libraries can be handed in, as we cannot gurantee proper sample handling within this period.

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Date: 16. October 2017

Subject: New Ordering System

Dear NGS user,

Today, we have been launching our new ordering system: Max Planck Genome Centre Cologne – Sequencing Order System, short MPGC-SOS. You will find it at https://mpgc-sos.mpipz.mpg.de . The old NGSOrderDB will be discontinued and is from today on only available to finish on-going projects. All new projects from today on will be managed viea the new system. If you have questions, do not hesitate to contact us.

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Date: 11. October 2017

Subject: Public Holidays in NRW in October/November

Dear NGS user,

This year is “Luther year”, so even catholic Northrhine Westphalia will celebrate Tuesday, 31st October as a public holiday in addition to “Allerheiligen” on the 1st November. As a consequence, the Genome Centre is closed both days and can not receive samples. On Monday that week (30.10.2017), the sample receiving capacity is extremely low. So please do not send samples to arrive before Thursday of that week.

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Date: 27. September 2017

Subject: PacBio control DNA in PacBio data

Dear NGS user,

To ensure the data quality on the PacBio Sequel, Pacific Biosciences offeres a control library to be sequenced with the normal sample. We have started to include this control library to be able to ensure best performance. However, some reads of your dataset may originate from this spike in. Please find the fasta file for download here .

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Date: 1. September 2017

Subject: Lisa is back, MiSeq, additional sequencing conditions

Dear NGS user,

A few things changed on the 1st of September:


Lisa Czaja-Hasse is back from maternity leave

Lisa Czaja-Hasse was gone for a year and is now back at her position, so you can contact her instead of Janina Fuss for all types of inquries. Or you could use our new, general e-mail address mpgc@mpipz.mpg.de for inquiries.

The Illumina MiSeq retired on 1st September

Due to very unsatisfying results on most MiSeq runs due very long standing Illumina chemistry problems and a consequently low user request for MiSeq runs with 2×300 bp, we decided to shut down the MiSeq instrument. We will finish the ongoing projects but no new projects will be accepted from 1st September. We are very sorry to do so. In case you want to sequence amplicons, please contact us to discuss alternatives on the HiSeq2500 with 2×250 bp.

New run mode(s)

We now have the possibility to offer addtional run modes: 2×75 bp on the HiSeq3000 and 2×150 bp/2×100 bp on the HiSeq2500 Rapid mode. You can request all run modes as Gb-amounts. However, the waiting times might be longer (up to eight weeks for sequencing) for the HiSeq2500, as we will have to fill the runs. The Hiseq2500 is recommended by 10x Genomics for sequenicng de novo projects and it has a non-patterend flow cell compared to the HiSeq3000. You can find the prices for those run modes here .

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Date: 17. July 2017

Subject: New prices, input amounts, instruments

Dear NGS user,

We now offer a range of new services that were tested here since Spring of 2017.


New RNA library prep protocol established – new input amounts for all kits

We have introduced a new library preparation protocol for RNA library preparations, which allows us to reduce the minimal input for RNA libraries with polyA enrichment and with rRNA depletion down to a 10 ng. We also provide now a minimum (lowest limit of each kit) and a recommended input amount, which ensures highest sequence diversity. If anyhow possible, follow our recommendations as:

A) For sequencing, more input will always result in a more complete (diverse) picture of the genome and a more reliable snap shot of the transcriptome at sampling time.
B) For (quantitative) comparative sequencing approaches, the same input amount must has to be used for all samples to ensure comparability! So please indicate “comparative gene expression analysis” for comparative RNA approaches as aim of project and keep in mind, that the input we use is then adjusted to the sample with the lowest input amount. In case you use DNA as template, indicate the need for comparative quantification in the comment field.
C) For any comparative approach, it is best practice to go for “PCR-free” library construction, if enough sample (min: 500 ng, recommended: 1500 ng) is available.


New instrumentation for long range sequence information available

PacBio SEQUEL

We have established sequencing with the PacBio Sequel system to replace the RSII, which increases the output per SMRT Cell significantly at comparable read lengths. All new projects are sequenced on the Sequel. The output per SMRT Cell has increased to aon average of 3 Gb (~2-6 Gb). However, we can only guarantee this output when one library is sequenced several times. PacBio recommends titrating of the optimal loading concentration for each library and estimates 3 SMRTCells to determine optimal loading concentrations. When only one SMRTCell is ordered, it will most likely not be loaded optimally and therefore the result might be below average.
In case you need less data or already have data created on the RSII, please contact Bruno Hüttel us for further advice. The RSII and the Sequel use different post run processing pipelines (SMRTLink and SMRT Analysis Suite), so data generated from both has to be analyzed separately until obtaining error corrected fastq/fasta files.
We will no longer offer the large AND extra large libraries for DNA, only libraries > 10 kb. The Sequel loading is not as size sensitive as the one for the RSII, so we can load broader size ranges and do not need to size the libraries anymore.
The full transcriptome can be sequenced on one SMRTCell due to the reduced size sensitivity. Therefore, we changed our IsoSeq libraries as well to one price for a full IsoSeq library with the option to size for larger fragments in addition.

10X GENOMICS linked read sequencing

The 10x genomics chromium controller allows for linked read sequencing of genomic DNA. This means, each fragment of high molecular weight DNA is used as a template in an isothermal amplification within an emulsion reaction, also including one of 4.2 mio individual barcodes. With the help of these barcodes, each read from an standard Illumina library containing these barcodes (GEMs) can be traced back to the strand of DNA it came from. This means, depending on your fragment size, you can gain location information over 50 -100 kb with short read sequencing. This is very useful for haplotype phasing, re-arrangement detection, de novo genome sequencing and genome polishing. It is especially suitable for genomes > 200 Mb, optimal for genomes > 1 Gb. If you want to know how this is done, please refer to this very nice, introductory video and if you want to know what else can be done, please visit the 10x Genomics homepage. At the moment, the set-up and software are designed for human and human-like genomes concerning ploidy, heterogeneity and phasing. It is possible to work with other genomes as well, but might require some further optimization.
To request for 10x linked reads, please chose 10 x linked read library in the NGS Order DB. Select 2 × 150 bp as run conditions, as this is the only supported read length for 10x libraries. We also offer a special bioinformatics package for linked read libraries to help with assembly, phasing and variant calling. For further questions regarding read length, feasibility and input requirements, please contact Janina Fuß.

femto Pulse for ultra low input and high molecular weight quality control

The femto Pulse is the successor og the fragment analyser, measuring quantities in the fg range and fragment sizes larger than 150 kb due to its pulse field function. Together with the PacBio Sequel and the 10 x Genomics, the femto Pulse completes the expanded field of high molecular weight DNA sequencing.


New services and quantities available due to frequent requests

We often have requests for additional treatment of samples, including measures to increase quality of the input, but also the extraction of optimal input DNA for different types of sequencing including high molecular weight DNA. We have added a range of the most common requests to our standard procedures. You’ll find details here.

We have added the possibility to request for full lanes on our HiSeq 3000 now, so please indicate this in the comment field. For non-standard read lengths, there is always the option to request a full run. We have standard prices now for 50 bp – single read sequencing on the HiSeq2500 and 3000, prices for all other read lengths will be made available on request.
These are on average slightly cheaper than requesting Gb or reads, but we do not guarantee output on lanes or flow cells! So we will pool the libraries for this lane equimolarly and in case the libraries differ in performance, we will not automatically re-sequence those. Illumina quality criteria apply for the definition of a failed lane/flow cell.

We have stopped offering MiSeq runs by the Gb mode, we only offer to sequence libraries in full flow cell mode at the Illumina list price. We will follow your request of loading concentration and PhiX spiking. If you do not provide specific instructions, we will follow Illumina’s recommendation and start with 12-14 pM loading concentration and 40 % PhiX spike-in. Please be aware that Illumina doesn’t guarantee quality specifications on the MiSeq runs with (16s/18s) amplicons, so the data output will be dispatched as it is, independent of quality and quantity.


All details on prices you will find here, while you can find information on the new instrumentation here , and the new loading concentrations in addition here.

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Date: 15. December 2016
Subject: Limited sample receiving capacities during Christmas time

Dear NGS user:
We wish a merry Christmas time to all our customers and collaborators!
Please note: From 23.12.2016 until 02.01.2017 NO new samples or libraries can be handed in. The absolute last date for sending in samples will be the 21.12.16 for everyone not handing in the samples personally!

At any timepoint later than that, we can’t gurantee the proper handling of the samples.

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Date: 01. September 2016
Subject: Updated Price List for NGS

Dear colleagues,

The MP-GC has updated the price list for next generation sequencing, new prices are valid for all projects created in NGS OrderDB since 01. September 2016.

Please find below a short summary of important changes in the next paragraph. For more details, please check the new price list here.

Due to optimizations in library preparation protocols, we were also able to reduce the minimum input material for some preparations significantly here.

Major changes for Illumina sequencing technology are:

• We have reduced our pricing per Gigabase for the HiSeq systems by 30 %.
• We were able to optimize the PCRfree library preparation protocol for DNA libraries, so we can offer PCRfree libraries at the same price as standard DNA libraries. The required DNA amount is still higher (2,5 µg DNA / sample).

Major changes for PacBio sequencing technology are:
• We observed a significant increase in read length and read numbers when performing damage repair for all PacBio libraries above 10 kb size, so we adopted this as a standard.

For comparison with other sequencing offers, please keep in mind that our prices are “final” prices for all Max-Planck members, no VAT (MwSt) will be added.

Please contact us in case of questions!

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Date: 23. June 2016
Subject: Example for full length transcriptome PacBio sequencing

cDNA Library Enrichment of Full Length Transcripts for SMRT Long Read Sequencing
The utility of genome assemblies does not only rely on the quality of the assembled genome sequence, but also on the quality of the gene annotations. The Pacific Biosciences Iso-Seq technology is a powerful support for accurate eukaryotic gene model annotation as it allows for direct readout of full-length cDNA sequences without the need for noisy short read-based transcript assembly. We propose the implementation of the TeloPrime Full Length cDNA Amplification kit to the Pacific Biosciences Iso-Seq technology in order to enrich for genuine full-length transcripts in the cDNA libraries. We provide evidence that TeloPrime outperforms the commonly used SMARTer PCR cDNA Synthesis Kit in identifying transcription start and end sites in Arabidopsis thaliana. Furthermore, we show that TeloPrime-based Pacific Biosciences Iso-Seq can be successfully applied to the polyploid genome of bread wheat (Triticum aestivum) not only to efficiently annotate gene models, but also to identify novel transcription sites, gene homeologs, splicing isoforms and previously unidentified gene loci.
Link publication

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Date: 14. December 2015
Subject: Limited sample receiving capacities during Christmas time

Dear NGS user:
We wish a merry Christmas time to all our customers and collaborators!
Please note: From 23.12.2015 until 04.01.2016 NO new samples or libraries can be handed in. Please do not send any samples within this period!

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Date: 30. November 2015
Subject: MP-GC services 2016/2017

Dear NGS user:

Within the last weeks several colleagues from different MPI`s contacted us with concerns that the MP-GC has to close its service soon. This information is really wrong! The MP-GC has been evaluated and it’s budget been proven until end of December 2017. So there is no question that we provide the service according to this application. All MPI scientists are highly welcome to use our services and for sure there is no need to change to any commercial service provider now.

We have presently implemented the new HiSeq 3000 sequencing platform, providing a much higher capacity, been much faster in running time, offering even increased quality compared to older Illumina systems and last not least pushing the standard read length up to 150 bp.

If you have any further question, please feel free to contact us!

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Date: 15. November 2015
Subject: Updated Price List for NGS

Dear colleagues,
The MP-GC has updated the price list for next generation sequencing, new prices are valid for all projects created in NGS OrderDB since 15. November 2015.

Some price correction were necessary, to adopt to reality, as we had a drastic increase in spring 2015 due to changed US-Dollar/Euro conversion. Nevertheless, most of the Illumina prices have been even reduced, because of the installation of the new powerful HiSeq 3000 system. Following, you will find a short summary of important changes, for more details, please check the new price list here.

For comparison with other sequencing offers, please keep in mind that our prices are “final” prices for all Max-Planck members, no VAT (MwSt) will be added.

Major changes for Illumina sequencing technology are:
• We now offer sequencing on the HiSeq 3000 platform! This new instrument generates longer reads (150 bp) in shorter time.
• We introduce routinely 2 × 250 bp paired end sequencing in so called “rapid mode” on HiSeq 2500.
• Our pricing scheme for Illumina sequencing has been modified from a read based system (counting number of reads) to a volume based system (counting number of Gbases). This will allow easier comparison with other projects, simplify ordering of a specific data coverage in Gb and last not least, stands for higher transparency. Consequently, we have calculated one unique price per Gbase for Illumina HiSeq sequencing at fixed 70 €/Gb.
• 150 bp read length will become the new standard and is also fixed to 70 € per 1 Gbase for single and paired end reads. Ordering 100 bp reads is limited to the next three month to finalize or fill-up on-going projects. For the future, we need to get high order-volumes of identical read length to fill up the HiSeq 3000 flow-cell a.s.a.p., therefore, we need again mainly one unique read length.
• Re-sequencing for matching the requested data volume, is the limiting situation for short project run times. To reduce project run times, we have opened the tolerance window for projects to be judged as “done” from 15 % to 30 %. Regarding costs, we stick to the same rules as before: only the generate data amount (within the tolerance window) will be charged. If more data is generated, only the ordered volume will be charged, nothing more. So it’s still easy to sum up the max. costs for your project, when ordering. Consequently, you might increase your order volume to get enough data done within the first run. Regarding the cost reduction, we think this is a time-saving and economically reasonable approach for all parties.
• Following frequent requests, mate pair library preparation has been added to our list of service offers.

Major changes for PacBio sequencing technology are:
• Due to increased demand, we now offer genomic DNA clean-up service.
• Full length transcriptome sequencing is now supported in a full service package. For details, please refer to the price list.

Please contact us in case of questions!

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Date: 18. June 2015
Subject: 2 × 250 bp sequencing on HiSeq2500 platform

Dear NGS user:
Upon request, we now also offer 2 × 250 bp sequencing on our HiSeq2500 platform. Please note: sequencing in this read length can only be performed in a rapid run mode with reduced capacities of 2 lanes per run. Currently, the minimal order quantity is 1 lane (approx. 140 million reads). For further information, please contact us!

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Date: 1. June 2015
Subject: 150 bp sequencing not offered any more

Dear NGS user:
We no longer offer 150 bp sequencing on the HiSeq2500 platform. Alternatively, 250 bp sequencing will be available soon.

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Date: 15. May 2015
Subject: PCR-free DNA libraries

Dear NGS user:
Due to many requests within the last weeks, please find here further information regarding PCR-free library preparation.

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Date: 5. February 2015
Subject: Limited sample receiving capacities during carnival time

Dear NGS user:
Please remember: From 12. – 18. February 2015 there is carnival season in Cologne!
We HIGHLY discourage from sending any samples within this period!

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Date: 9. January 2015
Subject: 454 sequencing no longer offered on regular basis

Dear NGS user:
Due to the rapidly decreasing number of requests throughout 2014, in 2015 454 sequencing will no longer be offered on a regular basis. Nevertheless: If you have a specific project request requiring 454 sequencing, please contact us to discuss what we can do for you!

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Date: 26. November 2014
Subject: Limited sample receiving capacities during Christmas time

Dear NGS user:
First of all: We wish a merry Christmas time to all our customers and collaborators!
Please note: From 20.12.2014 until 04.01.2015 NO new samples or libraries can be handed in. Please do not send any samples within this period!

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Date: 22. September 2014
Subject: Update of NGS OrderDB requires emptying browser cache

Dear NGS user:
We recently updated NGS OrderDB to make it more convenient for you. This update makes it necessary to empty the cache of your browser before starting the updated appliance. Please click here for guidance on how to do so.

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Date: 2. September 2014
Subject: Updated price list for NGS sequencing

The MP-GC has updated prices for NGS sequencing!
The new price list is valid for all projects created after 2nd September 2014 and can be found here.

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Date: 11. August 2014
Subject: Welcome our new colleague

The MP-GC welcomes a new colleague:
Last week our new bioinformatician Palani Kannan Kandavel started working at MP-GC. After an initial training phase, he will support the MP-GC with data analysis.

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Date: 21. July 2014
Subject: Welcome our new colleague

The MP-GC welcomes a new colleague:
Last week our new secretary Gabriele Breun started working at MP-GC. For general requests she will be available on daily basis between 10 am and 14 pm.

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Date: 11. July 2014
Subject: Further delay of 250 bp paired end sequencing

Dear NGS user:
Unfortunately, the recent problems with the MiSeq platform (see last entry) have not been solved so far. After the 3rd (!) repair, the device still is not free from defects! We hope this problem can be solved within the next days and will continue with routine 250 bp paired end sequencing as soon as possible.

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Date: 24. June 2014
Subject: Delay of 250 bp paired end sequencing

Dear NGS user:
Due to recent technical problems with the MiSeq sequencing platform all current 250 bp paired end sequencing requests will be delayed. The device has been in repair last week and currently is in repair again. We hope this problem can be solved rapidly and will continue with routine 250 bp paired end sequencing as soon as possible.

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Date: 25. April 2014
Subject: Alteration of sequencing read length

Dear NGS user:
Because of recent technical sequencing problems on the HiSeq2500 platform MP-GC has to reduce the standard sequencing read length from 100 bp to 98 bp for the next future.
For your understanding: MP-GC routinely uses 8 bp instead of 6 bp indices (as intended by Illumina) to increase multiplexing of libraries. This on the other hand reduces the possible read length by 2 bp. In the past we found a way to avoid this shortage by 2 bp, which is not supported by Illumina and which currently does not work anymore.
We are currently developing a strategy to get back to a 100 bp standard read length, but this will take some time. If the shortage by 2 bp causes problems to any of your ongoing projects please contact us to discuss how we can help to bioinformatically solve this problem.

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Date: 17. April 2014
Subject: Happy Easter!

The MP-GC team wishes happy Easter holidays to all customers and collaborators!

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Date: 3. April 2014
Subject: Automated upload of large projects into NGS OrderDB

Dear NGS user:
To save time when creating large (> 25 libraries) sequencing projects you now have the opportunity to fill out a corresponding excel sheet and send it to us for an automated upload into NGS OrderDB. For details click
here.
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Date: 11. March 2014
Subject: Illumina 150 bp reads no longer offered as standard service!

Dear NGS user:
Our standard sequencing read length for the HiSeq2500 platform are 100 bp and since March 2013 also 150 bp in rapid mode. After one year experience, we have to notice, that 150 bp sequencing at MP-GC has been used only quite rarely. As the rapid mode is associated with high additional time-consuming efforts, we will consequently no longer offer 150 bp sequencing in rapid mode on the HiSeq 2500 system. Nevertheless: If you have a specific project request requiring this read length, please feel free to contact us for help.