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Old 01-14-2015, 07:21 AM   #21
jwfoley
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I know nobody here cares about microarrays (nor should we?), but I think Illumina deserves some serious mad-scientist points for the NextSeq 550. If I'm understanding correctly, they've made a microarray adapter that fits in the flow-cell slot, and are reading it with the same scanner. More innovation than I'd expect from a company with a secure monopoly.

Maybe this is an attempt to wean the last few stragglers off arrays and get them into sequencing?
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Old 01-15-2015, 04:20 AM   #22
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Can these new flow cells still be overloaded, or has Illumina created the ideal clustering procedure? Just load your library at high concentration for maximal occupance of the flow cell "wells" and skip qPCR and titration.....
Anything known about the clustering procedure yet?
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Old 01-15-2015, 06:22 AM   #23
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Remembering back to the HiseqX announcement the little holes that now hold the clustering oligos (which is why this is a "patterned" flowcell) allow 1 oligo to overpower any others that try to cluster in there. I suppose you could somehow get it to overcluster but it wouldn't be the same kind of overclustering we are used to.

Clustering still appears to be on the cBot.

Here's some back-of-excel calcs I did on prices posted for the new 3k/4k reagents. Throughput assumptions are made based on average numbers given by Illumina in their specs and 8-lane FCs.

Screen Shot 2015-01-15 at 9.19.09 AM.png
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Old 01-15-2015, 06:53 PM   #24
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Quote:
Originally Posted by HeinKey View Post
Can these new flow cells still be overloaded, or has Illumina created the ideal clustering procedure? Just load your library at high concentration for maximal occupance of the flow cell "wells" and skip qPCR and titration.....
Anything known about the clustering procedure yet?
They still need to be quantified. Assuming similarity of HiSeq 4000 flow cell to HiSeq x, they can be overloaded which will reduce the reads %PF. Low loading will increase the number of duplicate reads. Illumina manual also states that underloading can reduce or result in unacceptable %PF. Loading is depend on library type (Nano or PCR-free).
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Old 01-16-2015, 07:07 PM   #25
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No rapid mode is a bummer for HiSeq 4000. I hope they can add that later.

I also want an upgrade of MiSeq
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Old 01-18-2015, 03:19 AM   #26
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Looks like there will be a V2 chemistry coming soon for NextSeq. Illumina claimed that the error rate can be on par with MiSeq/HiSeq. Hope this is true. This can be a nice upgrade for MiSeq.

HiSeq 4000 is quoted at $900K and 2500 is reduced to $690K. Both are more expensive than I expect.
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Old 01-20-2015, 01:01 AM   #27
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Quote:
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#SNPsaurus:
I assume it will be 2x 150 bp in 3.5 days.
2.1 to 2.5 billion clusters per flow cell, in a 2x150 bp run is 630 - 750 Gb.
With a speed >200Gb per day a 2x 150 bp run will take 3.5 days.

I wonder if we can upgrade the 2500 V4 machine to a 4000 version...
I asked Illumina and was told not. Apparently the cameras are more sensitive.

Also, the NextSeq550 capability for arrays is currently limited to the methylation and karyomapping chips, not sure why. They have suggested they may look into allowing iSelect if enough users are interested. Personally, I feel it would be a good move - core labs could use the NextSeq as a stopgap for priority NGS/genotyping runs if the HiSeqs/iScans are at capacity or down for repair.
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Old 01-21-2015, 10:36 PM   #28
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http://www.illumina.com/systems/next...ncer/kits.html

I see NextSeq V2 kits here claiming better error rate than v1. Is it true?

The throughput is the same??
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Old 01-22-2015, 05:48 AM   #29
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just visited an Illumina meeting in Brussels.

Current HiSeqs are not upgradable to the 3000 or 4000 versions. Hardware is too different between the systems.

4000 and 3000 can only sequence Human DNA with certain fragment length. WGS, exome and RNAseq will work well, other library types are not supported due to the limitations of the new exclusion amplification kinetics.

New NextSeq V2 chemistry was presented. New dyes in the V2 kits with much less spectral overlap compared to the V1 dyes. Discrimination between dyes is much better resulting in higher quality reads and lower error rates. Now comparable with their four dye chemistry.
All in all very informative again.
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Old 01-22-2015, 06:50 AM   #30
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Quote:
Originally Posted by HeinKey View Post
Current HiSeqs are not upgradable to the 3000 or 4000 versions. Hardware is too different between the systems.
No surprise there.

Quote:
Originally Posted by HeinKey View Post
4000 and 3000 can only sequence Human DNA with certain fragment length. WGS, exome and RNAseq will work well, other library types are not supported due to the limitations of the new exclusion amplification kinetics.
I highly doubt 4000/3000 are going to be limited to human samples only (DNA with certain fragment size perhaps because of ordered flowcells).
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Old 01-22-2015, 09:27 AM   #31
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From: http://www.illumina.com/systems/hise...lications.html

Quote:
The HiSeq 3000/HiSeq 4000 Systems deliver the highest daily throughput and lowest price per sample for multiple applications, species, and sample types, providing an ideal solution for production-scale sequencing labs.
Looks like no restrictions on sample types for 3000/4000.
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Old 01-22-2015, 11:32 AM   #32
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Quote:
Originally Posted by HeinKey View Post
#SNPsaurus:
I assume it will be 2x 150 bp in 3.5 days.
2.1 to 2.5 billion clusters per flow cell, in a 2x150 bp run is 630 - 750 Gb.
With a speed >200Gb per day a 2x 150 bp run will take 3.5 days.

I wonder if we can upgrade the 2500 V4 machine to a 4000 version...
Dumbass Question Alert, but does 2.1 billion clusters = 2.1 billion reads? Or would that be 2.1 billion clusters and with PE be 4.2 billion reads?

I guess my real question is on the HiSeq3000, how many reads do I get with 2x150bp PE?

Thanks in advance...
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Old 01-22-2015, 12:00 PM   #33
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Dumbass Question Alert, but does 2.1 billion clusters = 2.1 billion reads? Or would that be 2.1 billion clusters and with PE be 4.2 billion reads?
Yes. That is how illumina counts them.

Quote:
Originally Posted by cement_head View Post
I guess my real question is on the HiSeq3000, how many reads do I get with 2x150bp PE?

Thanks in advance...
Here are published specs (I assume you are interested in total sequence rather than number of reads): http://www.illumina.com/systems/hise...fications.html
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Old 01-22-2015, 11:47 PM   #34
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Our sales rep claimed there are no single read cluster kits available (yet) for HS3000/4000. Can anyone confirm? Sounds like a strange decision (if true) to me.
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Old 01-23-2015, 04:35 AM   #35
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Our sales rep claimed there are no single read cluster kits available (yet) for HS3000/4000. Can anyone confirm? Sounds like a strange decision (if true) to me.
That probably means "for the time being" ... as manufacturing is ramped up for new reagents/flowcells. Specifications list a 1x50 run, so single end kits may be coming in future (http://www.illumina.com/systems/hise...fications.html).
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Old 01-23-2015, 06:14 AM   #36
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Quote:
Originally Posted by GenoMax View Post
Yes. That is how illumina counts them.



Here are published specs (I assume you are interested in total sequence rather than number of reads): http://www.illumina.com/systems/hise...fications.html
No, I'm interested in total number of reads to calculate coverage.

So, what this (thread) is saying, is that for the same amount of money on a HiSeq2500 RR, if I did the run on a HiSeq3000 - I can potentially get >10x the amount of reads?

HiSeq2500 360M reads

HiSeq3000 4.2B reads

Have I got this right? If so, this is amazing!
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Old 01-23-2015, 06:22 AM   #37
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That's not really so amazing. A HiSeq 2500 in High Output mode will give you more than 6X as many reads (2 billion is the spec for v4). Rapid Run isn't for people who care about cost-effectiveness.
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Old 01-23-2015, 09:38 AM   #38
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My understanding is the the HiSeq 4000 per lane cost will be similar to the per lane cost of the HiSeq 2500. At our local facility, the per lane cost is similar for high-output or rapid run on the HiSeq 2500. So I would say roughly the same costs for:
HiSeq 2500 high-output 200M reads per lane (8 lanes per run)
HiSeq 2500 rapid run 180M reads per lane (2 lanes per run)
HiSeq 3/4000 320M reads per lane (8 lanes per run)
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Old 01-25-2015, 09:43 AM   #39
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There should be an exact max of clusters for HiSeq 3000/4000 defined by the number of microwells (325M as stated above?).
We're getting regularly 300M reads on the upgraded 2500. If 325M is correct, the increase is not very pronounced if the shortened run durations are not countered in.
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Old 01-25-2015, 10:05 AM   #40
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Quote:
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There should be an exact max of clusters for HiSeq 3000/4000 defined by the number of microwells (325M as stated above?).
We're getting regularly 300M reads on the upgraded 2500. If 325M is correct, the increase is not very pronounced if the shortened run durations are not countered in.
Are you sure you are comparing the same numbers (read, clusters, lanes, flow cell)? And for the HiSeq 2500 numbers are you talking about Rapid Run or V4 High Output?

The HiSeq 3000/4000 specs are 325M clusters per lane, which would be 650M reads per lane for paired end runs (using Illumina's method of counting).

The HiSeq 1500/2500 specs are 250M clusters per lane for V4 High Output (500M reads per lane). Our experience is pushing even slightly past 280M clusters per lane results in dramatic decreases in %PF and read quality.
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