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Old 06-18-2011, 03:58 AM   #21
mucku
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I found these webinars of IonTorrent very informative.

They also show data from the Broad Institute and others...

http://kevin-gattaca.blogspot.com/20...-training.html

As I remember the pH sensor doesn't really have a homopolymer problem... I think a hexamer is still called with a Phred 20 score. So I think part of the actual error might be not from the chemistry but from polymerase slippage during library preparation... Also the quality at position 100 is still extremely good, and you probably don't have to cut back so much of your reads because of low quality ends.
The PGM has really potential, I hope that not all of it is marketing...
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Old 06-19-2011, 04:49 PM   #22
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Look at real data before you state it doesn't have a homopolymer problem. It really, truly does -- it isn't something that can't be overcome, but the instrument+software has to date had difficulty enumerating homopolymers, even ones shorter than 6.
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Old 06-19-2011, 09:53 PM   #23
mucku
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Quote:
Originally Posted by krobison View Post
Look at real data before you state it doesn't have a homopolymer problem. It really, truly does -- it isn't something that can't be overcome, but the instrument+software has to date had difficulty enumerating homopolymers, even ones shorter than 6.
Sorry if it came across that I stated it doesn't have any problems... I have to apologize.
At this point there is so much competition in that area that probably only half of the information actually fits the systems and the other half are optimistic forward looking statements...
As I said I hope it is not all marketing...
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Old 07-08-2011, 08:48 AM   #24
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Default Real Comparison

I hardly tried to find some papers comparing that technologies but I have not been successful. Do someone knows some review, or paper showing the best characteristics of all these machines (IonTorrent PGM, Ilumina miSeq and Roche 4545Junior). As far as I see, here all the discussions are about what providers say in the catalogue?
Thank you very much
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Old 07-08-2011, 08:59 AM   #25
aleferna
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Try this one:
"Field guide to next-generation DNAsequencers"
amazing review

Molecular Ecology Resources (2011) doi: 10.1111/j.1755-0998.2011.03024.x
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Old 07-08-2011, 09:08 AM   #26
amascarell
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I read the abstract but unfortunatelly from my university I have no access...I'll have to ask for it somewhere.
Thanks for the advice.
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Old 07-09-2011, 06:28 AM   #27
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Quote:
Originally Posted by amascarell View Post
I read the abstract but unfortunatelly from my university I have no access...I'll have to ask for it somewhere.
Thanks for the advice.
http://molbiol.ru/forums/index.php?a...post&id=112976

http://molbiol.ru/forums/index.php?a...post&id=112978
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Old 07-09-2011, 09:20 AM   #28
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Thank you very much genseq...you safed my life
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Old 07-15-2011, 05:33 AM   #29
BadDNA
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Default MiSeq vs. Ion Torrent vs. GS Jr. vs. FLX+ vs. PacBio...

Updated tables that compare all of the currently commercially available & announced upgrades for Next Gen platforms are available at:

http://www.molecularecologist.com/next-gen-fieldguide/

It has prices for the instruments & ancillary equipment, performance based on stated goals of upcoming releases, summary "grades" for each platform for specific uses, etc.
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Old 07-15-2011, 07:06 AM   #30
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Quote:
Originally Posted by BadDNA View Post
Updated tables that compare all of the currently commercially available & announced upgrades for Next Gen platforms are available at:

http://www.molecularecologist.com/next-gen-fieldguide/

It has prices for the instruments & ancillary equipment, performance based on stated goals of upcoming releases, summary "grades" for each platform for specific uses, etc.
I found the grades kind of subjective but otherwise very good information for comparing platforms.
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Old 07-18-2011, 05:37 AM   #31
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Quote:
Originally Posted by BadDNA View Post
Updated tables that compare all of the currently commercially available & announced upgrades for Next Gen platforms are available at:

http://www.molecularecologist.com/next-gen-fieldguide/

It has prices for the instruments & ancillary equipment, performance based on stated goals of upcoming releases, summary "grades" for each platform for specific uses, etc.
It has prices for the instruments & ancillary equipment, performance based on stated goals of upcoming releases, summary "grades" for each platform for specific uses, etc.[/QUOTE]

Very useful table.

Did you update the Ion Torrent PGM 314 reagent pricing in light of the chip itself dropping to $99?
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Old 07-29-2011, 09:02 PM   #32
penpen
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here's one paper of combining application of GAII x, FLX and ion
torrent. sequencing by GA IIx , FLX, and SNP validation(amplicon sequencing) by ion torrent......
Attached Files
File Type: pdf WebbMiller.pdf (706.0 KB, 111 views)
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Old 08-08-2011, 05:29 AM   #33
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Very good question! And l'd like to see some reviews on this topic
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Old 04-24-2012, 03:59 AM   #34
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Default Best Benchtop sequencer: MiSeq or PGM or 454 GS Junior?

Finally a performance comparison of MiSeq, PGM and 454 GS Junior by researchers not the vendors

http://www.nature.com/nbt/journal/va.../nbt.2198.html
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Old 04-27-2012, 06:05 PM   #35
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Quote:
Originally Posted by rnaseek View Post
Finally a performance comparison of MiSeq, PGM and 454 GS Junior by researchers not the vendors

http://www.nature.com/nbt/journal/va.../nbt.2198.html
expect the Miseq and 454 runs were performed by the vendors.
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Old 04-27-2012, 10:44 PM   #36
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Just to be completely accurate, we also performed the 454 Jr runs. When we did this study the MiSeq hadn't yet been released.
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Old 05-05-2012, 05:46 AM   #37
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Great paper Nick. Does anyone have experience with Low Amplification libraries on the MiSeq? Their protocol demands 10ul of library be diluted into 1ml of HT1 and this is further diluted 2:1 before loading so a 200 fold dilution to load. Best I can tell the MiSeq needs 300-600ul in machine for the cluster formation (mostly to prime the lines). I bet only 50ul are actually in the chip during clustering so most of this sample is lost. As a result we are having a hard time delivering less than 18 cycle haloplex to the instrument and we can know this many cycles is not good for even coverage.
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Old 05-05-2012, 06:14 AM   #38
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Quote:
Originally Posted by Nitrogen-DNE-sulfer View Post
Great paper Nick. Does anyone have experience with Low Amplification libraries on the MiSeq? Their protocol demands 10ul of library be diluted into 1ml of HT1 and this is further diluted 2:1 before loading so a 200 fold dilution to load. Best I can tell the MiSeq needs 300-600ul in machine for the cluster formation (mostly to prime the lines). I bet only 50ul are actually in the chip during clustering so most of this sample is lost. As a result we are having a hard time delivering less than 18 cycle haloplex to the instrument and we can know this many cycles is not good for even coverage.
I am with you on this. The cBot/HiSeq seems to waste quite a bit of library, but the MiSeq is even more extravagant.

That said, obviously you could skip the the final 2:1 dilution down to 10 pM, by starting at 1 nM, instead of 2 nM. And, since you only use 600 of the 1 ml of neutralized ssDNA, you could start with 6 ul of library, instead of 10 ul. Together that would allow you to back off 2 cycles on your PCR.

That said, there is still the issue that 18 cycles yields a theoretical maximum of 2^18X amplification. ~256,000X to get to 2 nM. That suggests your initial concentration of amplification-competent library molecules is on the order of 0.01 pM. That would be about 10-20 million library molecules/ul--ie, probably below 10 pg/ul. Is that what you expect? If not, it could be library construction or enrichment PCR needs to be optimized a bit.

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Old 05-06-2012, 09:15 AM   #39
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Quote:
Originally Posted by Nitrogen-DNE-sulfer View Post
Great paper Nick. Does anyone have experience with Low Amplification libraries on the MiSeq? Their protocol demands 10ul of library be diluted into 1ml of HT1 and this is further diluted 2:1 before loading so a 200 fold dilution to load. Best I can tell the MiSeq needs 300-600ul in machine for the cluster formation (mostly to prime the lines). I bet only 50ul are actually in the chip during clustering so most of this sample is lost. As a result we are having a hard time delivering less than 18 cycle haloplex to the instrument and we can know this many cycles is not good for even coverage.
The instrument actually takes 400 out of the 600 in the well as best I can tell from reading the recipe files...unfortunately the post run cleanup washes flush out that well so it's hard to estimate the dead volume...but I bet you could get away with less than 600.

For denaturing low concentration libraries (and all libraries for that matter), I've moved 100% to using NaOH followed by neutralization with HCl, followed by dilution with HT1. This removes the (somewhat ridiculous) need to dilute out the NaOH...and frees you to denature any way you want.

My preferred protocol is to dilute the library into 40ul of water or EB, add 1ul of 2N NaOH (to get to 50mM), incubate 5 min, and add 1ul of 2N HCl. then dilute this directly to whatever loading concentration I want....this works great and prevents having to denature a huge quantity of library.

Next on my list is to add ~10mM Tris to the HT1 to further smooth out any variations in pH (I think this is recommended in the Sanger optimization paper), but I haven't run into any problems yet.
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Old 11-08-2015, 12:45 PM   #40
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Pacbio is worth considering too. However if you are doing lots of MB/whole genome sequencing then Illumina is the most cost effective option these days? I've done a summary of the pros/cons here
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