Sequencing service:Things to know

[Melissa]

Hi, here's my experience when looking for sequencing service. I thought I should post it.

Things you should know:

1. Where to look for a service provider?
   Isn't this obvious, people? Go online!
   
   I can name 3 types of service provider: commercial company, Academic institutions/Genomics center and manufacturer company. Example of manufacturer's sequencing service is Illumina Customer Service Evaluation (cse) and FastTrack service. Commercial company is generally more expensive but flexible to customize your needs.
   
   You can choose a local or overseas provider. The local provider might be the middleman. I would say that the difference is you can have a tête-à-tête with the local provider while you can only discuss your project with provider abroad through emails. Also, let them know your budget so they won't suggest something extravagant.
   
   There's a sequencing service list available at http://www.nucleics.com/DNA_sequenci...e-reviews.htmlwhich refers only to Sanger sequencing but worth a look. Some of the labs might own NGS machines. Hmmm... maybe I can work out a list for the community.
   
   
2. Price
   Pricing is usually not listed in the website. So you need to contact them for the quote. There will be different pricings for academic/profitable user, external/internal user and location (country-based) etc. Don't forget the tax (if any).
   Beware of $ sign! Make sure you know which currency are they referring to. Online currency converter is very convenient and up-to-date.
   
   
3. Details
   You would wanna know
   • the amount of reads generated/ data (Mb)
   • read length
   • cost for libary construction, sequencing (/lane or /flow cell) and bioinformatics support
   • single/paired-end sequencing
   • multiplexing/barcoding
   • file format of the results
   
   
   They would wanna know
   • sequencing purpose
   • organism of interest
   • size of genome/transcriptome
   • availability of reference genome
   
   
   
   
4. Bioinformatics support
   Most of them would provide bioinformatics service unless they are very busy with some big sequencing projects. Most likely you will not have access to softwares that come together with the platform like Newbler and GApipeline. You can have the analysis done for you. If anything goes wrong with your sequencing, have them to re-sequence (preferably with no additional charges )
   
   
5. Courier service
   Find a courier company that can replenish dry ice for you during sample delivery if the delivery takes 2-3 days. You might worry about degradation problem if you send starting material in the form of total RNA. Dissolved your samples in RNAse-free water is ok.
   
   
6. Turnaround time
   How long does it takes to get the sequencing results?
   We won't wanna send our samples and never hear from them again. Hehe
   Well, it depends on the queue, your number of samples, type of sequencing and starting material. It usually takes ~ 8 weeks for Solexa and 454 sequencing which includes library construction and sequencing.

Hope this helps.

Melissa

[ECO]

Great first post for this forum, thanks Melissa.

[zee]

I've been looking at some service providers for sequencing. Some of the well established ones are:

WashU
NCGR

I did stumble upon a company who actually lists their sequencing pricing on their frontpage and they do 'car pools' to conserve sequencing lanes on the GAII. They even have a schedule showing the availability queue.
Perhaps more service providers could be more open with displaying this information that's helpful to the user.

[ECO]

Zee, thanks for being conscientious and not mentioning the company....but please feel free. My warnings about "commercial content" do not apply to discussion of companies by members....

You're talking about this company right? http://cofactorgenomics.com/ ??

I agree that their upfront pricing and scheduling is the BEST idea to hit genomic services in a long time. And it probably benefits them as well, reducing the need for a ton of customer service to deal with "feeler" requests.

[zee]

Spot on ECO. I'm in agreement with forums not becoming like saturated with advertisements.

Staying on topic though, I think there should be more transparency on outsourcing sequencing costs because it can vary greatly depending on Geographic location. An example would be Asia pacific, we could end up paying 50% more for a local sequencing job even if we sent samples overseas.

Therefore turnaround time and shipping cost/safety become a significant concern. Certain vendors in Asian countries may even increase their margin by 200% because the technology is so new in the region. But I suppose that's the business world.

How about a sequencing "Price Watch" for consideration? It could be done by geographic location if parties were willing to submit their data. And perhaps other parameters could be highlighted. I'm just thinking aloud.

[ECO]

Love that idea. I'll think about the best way to do it...we could probably go with a sticky post (similar to the massive bioinformatics thread)...

[frozenlyse]

One thing I would suggest you find out is how much experience the company has with the next gen sequencer they have - my service provider turned out to only have had their solexa for a few months so we got stuck with quite a few teething problems, and it took months for us to get results!

[zee]

One thing I would suggest you find out is how much experience the company has with the next gen sequencer they have - my service provider turned out to only have had their solexa for a few months so we got stuck with quite a few teething problems, and it took months for us to get results!

That's a good point. Our lab (also in Australia) also had that issue with a sequencing service provider. They had also done virtually no QC on the sequences before sending to us. It creates problems and I've learned not to make any assumptions that the data is clean, rather expect the worst and hope for the best.

[Melissa]

Totally agree. That's why it'll be important to get recommendation from those who have done it there. We will be using the service that has been used by our collaborator.

[zee]

Perhaps it's best to ask them for a couple of things e.g.

Intensity/Percent Caller per cycle number (for Illumina)
% Reads with low complexity
%Reads with average quality below X
Count of unique sequence tags
A plot of average read quality vs read length - we expect it to decline towards 3'
Any adaptor sequences that may be needed to strip off - certain mapping tools will do this 3'/5' stripping prior to alignment

[Melissa]

Addition:
And do make sure to ask the right person about the technical issues. If not, your emails will be forwarded, cc and lost in... space.

WARNING: Please follow all the advices above at your own risk!

[bioinfosm]

ANy other quotes from companies/groups for bioinformatics solutions on next gen?
They usually say mapping charges of $$$ per 100MB etc, or per lane of solexa, etc

[zee]

I'm aware of a company in Australia that charges between USD $5K-$6K per sample on a GAII. I think the AGRF is another institute that charges more.

[Ryan_Richt]

Hey Guys,

A disclaimer: I do represent Cofactor Genomics, but I can be impartial!

All of the tips outlined so far are great. A few more:

*Whether they quote sequencing in lanes or by the mega-base, what % of those reads are "pass filter" according to that instrument manufacturer's pipeline? If they quote by lane, how much pass filter data are you guaranteed?

*For transcriptome projects, can you send total RNA or do you need to make cDNAs yourself (and by which of many methods)?

*What happens if the run/library fails?

*Does the price include alignments, and using what software? Does that provide multiple-matches, none, only 1, or random placement in the case of ambiguity? How many mismatches (and at what quality?) are tolerated? Did they align the filtered data or all of the raw data?

*Beware of people, especially service providers using the word "assembly." A "reference-guided assembly" is not an assembly. Make sure to qualify your questions with "de novo" assembly if that is what you want.

*If you receive any analysis help (and I mean post-alignment/assembly) make sure the provider respects and uses mathematically sound algorithms and places an emphasis on probabilistic reasoning. Countless random scripts produce results that are not worth looking through. You can also conjure up loads of graphs, tables and statistics that are completely uninformative.


Also a few tips for USING a service provider:

*Protect yourself from the service provider trying to blame a failed run on you.

Start by sending high quality samples, *especially* if you starting with limited or degraded DNA like ChIP products or FFPE samples. Check your 260/280 & 260/230 ratios, look at the whole curve on a Nanodrop, run a quick gel, and maybe even run in on the Bio-Rad Experion or Agilent Bioanalyzer.

If you are doing certain extractions, you might end up with a lot of DNA or RNA masquerading as the other on say, the Nanodrop. This does not count towards the amount you need to send! DNAse and RNAse are pretty cheap and pretty mean/fast/complete, so its a good idea if you are unsure.

Email all of this when you mail your sample. Of course we trust you, but we will do it again anyway so expect a chat ;-).

If things are bad salt/metal/protein wise, try a Phenol/Chloroform extraction followed by EtOH precipitation with several washes.

You need certain fragment sizes for each platform, if you have big fragments, we can make them smaller. BUT if you fragment to some intermediate sizes, the method of choice might be unable to break them down further into the size range we need. Feel free to ask before you Sonicate/Shear/Nebulize/etc.

*Longer reads or mate-pairs, etc, are not necessary on every project. However, you __will__ be disappointed with the results (especially de novo assembly) if you decide to "try it anyway." As much as service providers will try to up-sell you unnecessarily, sometimes these upgrades are important. (and sometimes not!)

*If you are getting any kind of bioinformatics/computational biology help, please feel free to send any information you already have, as well as important evolutionary or public database information. We may not know there is a treasure trove website devoted to the particular sub-species you work on, but if we did, we would use it!

[ECO]

Wow, great post Ryan. Welcome!