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Old 07-09-2016, 12:13 AM   #1
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Default Long synthetic read ??


Is there anybody have already worked with Illumina Long read ?

If yes I would like your feedbacks

1) It is described to be long up to 10K; is it really the case ?

2) What is the error rate ? (Pacbio is 15%)

3) What's the amount of DNA required ?

4) Is it relevant in case of de novo assembly ?

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Old 07-09-2016, 08:08 AM   #2
Brian Bushnell
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Cross-posted here. Please include links to your other posts when you ask the same question in multiple forums, so people know if it's already been answered.
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Old 07-09-2016, 09:11 AM   #3
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However my 1st and 3rd questions still remain.
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Old 07-09-2016, 06:13 PM   #4
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#1 Individual reads are not 10K long. No current Illumina sequencers can produce reads that long. That is the starting length of DNA that goes into these libraries.

#3 Libraries can be created using as low as 500 ng starting DNA (info from the PDF application notes on the page you linked above).
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Old 07-10-2016, 07:08 AM   #5
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Illumina's (Moleculo) "Synthetic" long reads are assembled from short reads originating from highly multiplexed (nextera) sequencing libraries themselves generated from 8 kb to 10 kb long PCR product libraries, themselves generated from sheared and highly diluted genomic DNA samples.
I assume that with the advent of the 10X Genomics Chromium chemistry there will be very few applications for which the Illumina product will be preferable.
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Old 07-11-2016, 07:59 AM   #6
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I'm not sure the BioStars answers really captured what the pros and cons of the system were. The good part of the kit is the ability to create 384 libraries, each from less than 1 ng of DNA. Each well of the 384 well plate is intended to contain about 300 ten kb chunks of the genome of interest. About 3 megabases of the genome in ten kb hunks.

There are two main minuses, though.

(1) To get a good Spades assembly of one well, you want at least 30X coverage. So 0.1 billion bases of sequence per well. 384 wells -- that is around 40 billion bases of sequence. So, basically, a full Rapid flowcell for one set. What do you get? If all went perfectly, you would get 300x384 ~= 115 thousand 10kb "reads". Or 1.1 billion bases of "raw" sequence. They should be highly accurate. But imagine the number of lanes you need to go even 10x on a mammalian genome! You would like want HiSeq 3000/4000 lanes for this. And I'm not 100% sure the HiSeq3000/4000 could handle the goofy 8 base single indexes.

(2) The second issue is that I don't think the long PCR used to generate the 10kb templates for each well of library construction is low bias. Shorter templates are clearly preferred by the polymerase and so there are likely other biases as well.

Other applications that might be good for this kit:
How about full length cDNA assemblies? Some tricks to this, I'm sure, but it could work.

As far as the kit being "hard to use", I don't think that is the case. Illumina did a good job of laying it all out and none of the steps are particularly difficult. Were the prices of sequencing to drop ten-fold, I think this would be a much more attractive technology.

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