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Old 09-11-2011, 05:27 AM   #1
and.maz
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Default Preparing an Illumina Library

Dear Users,
I am sorry if this thread looks too simplistic or if it contains stupid questions.
The problem is that I am a neophyte of the field.
In short: I am a clinical cardiologist, involved in the research field of inherited heart disease.
I have the duty to prepare a panel of human genes related with channelopathies and cardiomyopathies to study with NGS (Illumina technology).
Some companies, like for instance Harvard partners, have already commercialized something similar.

My questions are the follows:
1. is there any practical guidelines I could read on the matter?
2. how many genes are OK to include in the library? (the number could range from about 40 if I choose the ones with the highest yield to more than 100 if I add also rare genes or candidate genes).
My doubt is whether it is better to study few genes supported by the strongest evidence or to scan also genes for which less is know. If the price / work for a largest panel is not too high, I would like to have all the possible information from my patients.
Additionally, how much would it cost to add new genes to my library?
I imagine that also the interpretation process would be more complex, if you study a very high number of genes.
3. Is there any gene which is not good to add to the library (for instance, titin is very large and apparently hard to study)?

thanks for your suggestions and your patience.
It would be very nice for me to see an Illumina machine working, on the field.

all the best.
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Old 09-11-2011, 06:24 AM   #2
Heisman
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How many individuals do you plan to sequence?
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Old 09-11-2011, 08:16 AM   #3
and.maz
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Uff... many. Lots of patients
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Old 09-11-2011, 08:31 AM   #4
Heisman
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Your best bet may be to do a targeted enrichment protocol. I have used the Agilent system, which you can read about here: http://www.genomics.agilent.com/Coll...ew&PageID=2094

I've heard from others that the new Illumina system works better, but I haven't tried it and I don't know what it costs. For the Agilent system, I think it's around $10,000 to order enough reagents to sequence 10 individuals if the protocol is followed directly. This does not cover the cost of sequencing. However, you can probably pull multiple individuals together for one experiment. There are a couple of papers indicating pulling 8 individuals together is doable. In that case you can get reagents for 80 individuals for under $15,000 (the most expensive reagent (RNA baits) would still be in the first $10,000 but you'll have to order more reagents to generate the extra libraries when pooling more individuals together). Again, this doesn't include the cost of sequencing.

Maybe others will have better ideas, but while this one costs a decent amount of money, it is not terribly time consuming (sample prep can be done in under 2 weeks, although it can take 7 weeks to get the reagents after ordering).
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Old 09-11-2011, 10:12 AM   #5
and.maz
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thanks. i will study the matter.
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Old 09-13-2011, 02:06 PM   #6
josdegraaf
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You want to look at genomic sequence? Or expression? You want to have whole genes or snps?
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Old 09-14-2011, 12:34 AM   #7
and.maz
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We want to look at genomic sequence in a clinical settings. We plan to scan all the exons in genes related to cardiomyopathies/channelopathies/sudden death syndrome. We look for pathological mutations, more than for SNPs. The SNPs we plan to add them to our DB, in order to store them for future studies.

Till now we make sequence study of selected genes (exons + closer intronic regions) with the Sanger method.

Is it a good explanation?

Thanks.
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Old 09-14-2011, 01:35 AM   #8
josdegraaf
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you can indeed do targeted capture, Agilent SureSelect, Nimblegen Seqcap EZ, Flexgen Flexselect are some companys that offer that. With Nimblegen atm being able to do pooling of different samples prior to capture (this could mean quite some reduction in costs) although this is also possible with the other two just not in a kit way as we speak.

You can also PCR your regions of interest up, barcoded and sequence those. Illumina will have something for that related to the Miseq but also the other sequencers will be able to handle that.

Concerning cost you would have to talk to the companies.

Cheers
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Old 09-14-2011, 02:45 PM   #9
NextGenSeq
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Personally I would just do whole exome capture.
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Old 09-15-2011, 12:42 PM   #10
james hadfield
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I'd recomend you look at Fluidigms Access Array. With this you can amplify 48 patient samples for up to nearly 500 exons or other loci. We have been using this for two years now. It allows you to PCR amplify and create libraries ready for sequencing. We are using 384 barcodes so you could get 3072 patient on a single flowcell. You can do a bidirectional sequencing which allows both ends of the PCRs to be read in a single end run. Cheap and fast!

You might also wait for Illuminas custom truseq based on GoldenGate, or HaloGenomics, or MIPs.

I prefer these methods as you do not need to make libraries and Fluidigm uses 50-100ng of DNA.
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