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Old 06-11-2012, 09:31 AM   #1
cumulonix
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Default 200-250bp read length

My application requires read lengths of 200-250bp, which looks doable with the 100bp and 150bp paired end reads on HiSeq and MiSeq. I was wondering
1) Is there another technology currently in the market with read lengths in 200-250bp region? I've tried the PGM 200bp kit which performed abysmally. Most reads had at least 1 read error, which is not suitable for my application.
2) If I use the illumina platform, how many # reads can I expect for the HiSeq and MiSeq? With paired end 150bp reads, what is the max length of the fragment with adapters?
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Old 06-11-2012, 12:03 PM   #2
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Originally Posted by cumulonix View Post
Most reads had at least 1 read error, which is not suitable for my application.
Could you explain this further? I am wondering which technologies (besides Sanger) will give fewer than 1 error in 200 on average.
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Old 06-12-2012, 05:35 AM   #3
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The other two current technologies in this read length space would be PacBio in CCS (Circular Consensus Sequencing) mode and 454. With the v2 chemistry, you may get a lot of 2-3Kb reads, so if you really have inserts of ~250, that would translate to 8-12 depth on each fragment.

For PGM, you could try their paired end protocol -- it is claimed to reduce the error rate substantially.
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Old 06-12-2012, 11:02 PM   #4
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Could you explain this further? I am wondering which technologies (besides Sanger) will give fewer than 1 error in 200 on average.
ah, my bad. I meant out of ~600k reads I got from the 200bp kit, 314chip 0 reads were at least 200bp long and contained no errors.

Some experimental detail here - I use sequencing to estimate relative growth rates of ~200bp alleles in yeast (library pooled and cultured in 1 flask). My test library in the torrent run had a diversity of 55 (Sanger sequencing verified). I could assign ~95% of the reads to my reference library (BLASTing the reads agaist the reference database), but the overall poor quality (avg length is 135bp, 0 reads cover the length I want sequenced without errors) makes me want to try another platform for my error-prone PCR library.

Last edited by cumulonix; 06-15-2012 at 07:24 AM.
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Old 06-19-2012, 11:28 AM   #5
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The MiSeq can run 200 to 250 cycles.
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Old 06-20-2012, 10:02 AM   #6
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You can easily get reads of that length on a 454. The now older Titanium chemistry will sequence your amplicon clear through with no problems whatsoever (assuming there aren't any homopolymers more than 5 or 6 bp long). Not every read will be perfect, of course, but at that length most will be and there are filtering strategies to get rid of those with errors.
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Old 06-20-2012, 10:29 AM   #7
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Sorry, bgold04, I was responding to the original poster, not to your post. I'm afraid I can't help you with your question.
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Old 06-20-2012, 10:43 AM   #8
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Well, the original poster said he had tried the PGM but wasn't happy with the results and asked whether any other technology on the market can get read lengths in the range he needs. 454 can, so that's a valid answer to the question. I mentioned it because, based on the description of the problem, it seems to me that 454 may be the best technology for this specific application, assuming the sequencing depth is sufficient to answer the question at hand.

Frankly, I'm a little confused as to why you're confused by this.
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Old 06-20-2012, 01:05 PM   #9
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Originally Posted by bgold04 View Post
I need to do a run MIT-Broad style (mixing two kits together) for about 300 cycles and get what I can get. I have no access to a machine, but I can supply a machine ready library and dollars. Where can I do this run for about $ 1000 (maybe more for the two kits) and get results? I am willing to go where-ever I must, or at least deliver the sample and money.
I'm assuming you want 300bp single reads (not 2x300). If this is the case, you don't have to mix kits...just edit the sample sheet.

Hopefully you can find a place to run it, if you run into trouble, shoot me an email.
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Old 06-20-2012, 05:42 PM   #10
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The OP said he needed 200-250bp perfect reads. I think he'll be disappointed with SBS as well.
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Old 06-21-2012, 06:36 AM   #11
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On the MiSeq you would get about 5 M reads. With the 2x150 kit your 200 bp alleles should not be a problem. About 30 to 50 % of the reads should be free of errors. However, with some scripting you should be able to also match the reads with errors.
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Old 06-21-2012, 07:59 AM   #12
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The OP said he needed 200-250bp perfect reads. I think he'll be disappointed with SBS as well.
Well, if all of the reads must be error free, then what he wants is impossible. No current technology is capable of providing that. I only have experience with 454, but in my experience, at 200-250 bp, he can expect up to 90% perfect reads with the Titanium chemistry on 454. I don't have experience with the other platforms, so I can't say anything about them.
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Old 10-02-2012, 03:37 PM   #13
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I've decided on using the HiSeq for my experiments based on 2 factors
1) Easy Sample prep: I have access to a core facility with the HiSeq. Sample preparation on the one-touch followed by Torrent Sequencing is relatively demanding (it might get easier with # trials, but I don't have the $$ to cross that activation barrier).
2) ~200 million reads from a PE lane: Apart from a high resolution on fitness, large read numbers for alleles lets me handle errors (>1000 counts for allele1 + < 5 counts for point_mutant_allele1 => ignore point mutant.. or, in the future, 'correct' base calling / synthesis errors)

There is another issue I need to resolve though...
The core facility in my university only does SE-50 and PE-100. I would like to attempt PE-150 or more. Is there a place I can ship my sample and get it sequenced?
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Old 10-03-2012, 09:06 PM   #14
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I don't think the hiseq can push out to 150bp. Not very well at least.
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Old 10-04-2012, 07:07 AM   #15
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At the moment, 150pb read are not supported by Illumina on the Hiseq2000.
I think it's still possible to run it on the machine but there is a strong probability that your error rate explode at the end of the read. And I don't think it will be suitable for your application.
Unofficially, our FSE told us that a "correct" error rate for a 100nt read was below 2% (I think it was V2 kits a that time, it may have vary with V3 kits). I won't be surprise if your reach 4 to 5% error rate with a 150nt read.
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Old 10-04-2012, 08:52 AM   #16
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Quote:
Originally Posted by huguesparri View Post
At the moment, 150pb read are not supported by Illumina on the Hiseq2000.
I think it's still possible to run it on the machine but there is a strong probability that your error rate explode at the end of the read. And I don't think it will be suitable for your application.
Unofficially, our FSE told us that a "correct" error rate for a 100nt read was below 2% (I think it was V2 kits a that time, it may have vary with V3 kits). I won't be surprise if your reach 4 to 5% error rate with a 150nt read.
Here is a plot of the error rate (phiX) for our most recent HiSeq2000 run:



Just for the top surface. Bubbles drive the error rate up higher for the bottom surface. Even so the error rate at 100 bases is generally below 0.5%.

The Sequence Analysis Viewer (SAV) shows >80% of the reads with no errors (counting both surfaces). Again, this is based on aligning phiX reads spiked into 7 lanes of the flowcell to phiX by the instrument software.

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Old 10-05-2012, 06:16 AM   #17
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I've checked our last 2*100nt run. On the PhiX control lane, error rate is between 0.8 and 1%. But for the spiked PhiX in our sample, it's just above 1%.
:'(
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Old 10-05-2012, 06:47 AM   #18
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MiSeq is now capable of 2x250b PE
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Old 10-05-2012, 09:52 AM   #19
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Also the upgraded MiSeq gives 16 million reads.
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Old 10-07-2012, 01:25 PM   #20
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Also the upgraded MiSeq gives 16 million reads.
16million clusters or reads?
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