MiSeq - Page 4

GW_OK · 05-27-2011, 02:40 PM

It's so helpful that Illumina put it's cluster density numbers up without telling anyone how many square millimeters a miseq flowcell has. Does anyone know the magic number?

05-27-2011, 02:55 PM

they said 3.5-5million clusters total

GW_OK · 05-27-2011, 02:56 PM

Huh, must have missed that.

james hadfield · 05-29-2011, 11:28 AM

There is also a PowerPoint presentation in PDF format on the Illumina site which gives more infomration on the poster.

One interesting thing is the error plots for all runs they show. Something I have not noticed before (need to look next week) is that all the plots show a kind of step-like profile increase in quality over the first 20 or so bases. Then there is a drop off in quality as run length increases. However this is not a simple drop as shown on slide 14.

Slide 18 shows HiSeq v MiSeq for 100bp runs of an E. coli library. Both show a drop in quality at about which recovers immediatley after, however it is at 75/65bp for Hi/MiSeq respectively. This may be an error in the library???

Slide 5 shows some lovely data on amplicon resequencing and mutation detection. It makes me wonder what the final reports are going to look like when this gets into a non-bioinformaticians hands?

raonyguimaraes · 07-09-2011, 07:22 AM

Just found this hilarious videos about MYSEQ and HISEQ

http://www.youtube.com/watch?v=GUr17pHezUo

http://www.youtube.com/watch?v=_xBwWMT7PgA

http://www.youtube.com/watch?v=gStCvyGpnRU

http://www.youtube.com/watch?v=G20F7GkHsmY

ShiveringFire · 07-20-2011, 12:11 PM

We have a barcoded/indexed library that requires custom sequencing primers to run on Illumina. Custom sequencing primers prevent sharing lanes with other projects. Perhaps Miseq could be an option since it is possible to use entire flowcell. Illumina webinar "my samples, my study, myseq" mentions @22nd minute about sample loading but does not say anything about custom primers:

http://www.illumina.com/events/webinars.ilmn

Can we use custom sequencing primers in MiSeq?

razibus · 07-21-2011, 02:13 AM

I think this service will be provided via GoldenGate platform.

ShiveringFire · 07-21-2011, 08:27 AM

Who has these machines? How can I locate a collaborator having one? Preferably around southeast US.

krobison · 07-22-2011, 07:51 AM

Given that GoldenGate is a technology upstream of their arrays, could you expand on how this is going to be applied to sequencing?

james hadfield · 07-22-2011, 08:10 AM

The Illumina GoldenGate PCR is used for lower plex genotyping and other applications on arrays. The PCR is likely to be modified such that a set of locus specific primers have regions with complimentarity to Illumina adapters. GoldenGate on arrays allows up to 3072 multiplex reactions. This offers the possibility of 96-384 and even up to 3072 loci (e.g. exons) being targeted by PCR in a standard 96 or 384 well plate from very low input DNA. And generating sequence ready amplicons which would fit neatly into a MiSeq PE run. The whole workflow including PCR could be completed in one day with results returned, analysed the next morning depending on run configuration.

This workflow would compete with other targeting approaches aimed at low multiplexing and not with whole exome resequencing for instance. As it could be 96 or 384 well based I could see a panel of cancer genes being resequenced at every exon as a standard product any user could buy from Illumina, add their samples to and get results on really quickly. In fact I've asked to do exactly that. The GoldenGate product was announced as being launched at the same time as MiSeq in September/October.

james hadfield · 07-22-2011, 08:13 AM

The Illumina GoldenGate PCR is used for lower plex genotyping and other applications on arrays. The PCR is likely to be modified such that a set of locus specific primers have regions with complimentarity to Illumina adapters. GoldenGate on arrays allows up to 3072 multiplex reactions. This offers the possibility of 96-384 and even up to 3072 loci (e.g. exons) being targeted by PCR in a standard 96 or 384 well plate from very low input DNA. And generating sequence ready amplicons which would fit neatly into a MiSeq PE run. The whole workflow including PCR could be completed in one day with results returned, analysed the next morning depending on run configuration.

This workflow would compete with other targeting approaches aimed at low multiplexing and not with whole exome resequencing for instance. As it could be 96 or 384 well based I could see a panel of cancer genes being resequenced at every exon as a standard product any user could buy from Illumina, add their samples to and get results on really quickly.

In fact I've asked to do exactly that. The GoldenGate product was announced as being launched at the same time as MiSeq in September/October.

james hadfield · 07-22-2011, 08:17 AM

GW_OK here is an answer from my measurements.

A MiSeq flowcell is 1.5x23mm see a picture here… Core-Genomics blog, however only a portion of this is imaged as far as I am aware.

razibus · 07-22-2011, 09:28 AM

Quote:

Originally Posted by krobison

Given that GoldenGate is a technology upstream of their arrays, could you expand on how this is going to be applied to sequencing?

Like James explained, Goldengate is a large platform which includes several technologies like primer design. This platform is also shared and used on other technologies from illumina, for example BeadXpress if I'm not wrong.

system7 · 08-30-2011, 07:06 PM

Illumina makes a strong statement regarding MiSeq and Ion Torrent data quality...

http://www.illumina.com/systems/mise...e=illumina.com

krobison · 08-31-2011, 06:13 AM

In addition to Illumina's analysis, there are analyses from Ion (referenced in my blog item) and from Edge Bio (which provides Ion as a service).

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07-20-2011, 12:11 PM	#66
ShiveringFire Junior Member Location: Athens, GA Join Date: Oct 2009 Posts: 7	Custom sequencing primers in MiSeq? We have a barcoded/indexed library that requires custom sequencing primers to run on Illumina. Custom sequencing primers prevent sharing lanes with other projects. Perhaps Miseq could be an option since it is possible to use entire flowcell. Illumina webinar "my samples, my study, myseq" mentions @22nd minute about sample loading but does not say anything about custom primers: http://www.illumina.com/events/webinars.ilmn Can we use custom sequencing primers in MiSeq?

07-21-2011, 08:27 AM	#68
ShiveringFire Junior Member Location: Athens, GA Join Date: Oct 2009 Posts: 7	MiSeq service Who has these machines? How can I locate a collaborator having one? Preferably around southeast US.

05-27-2011, 02:40 PM	#61
GW_OK Senior Member Location: Oklahoma Join Date: Sep 2009 Posts: 411	It's so helpful that Illumina put it's cluster density numbers up without telling anyone how many square millimeters a miseq flowcell has. Does anyone know the magic number?

05-27-2011, 02:55 PM	#62
SeqAA Guest Posts: n/a	they said 3.5-5million clusters total

05-27-2011, 02:56 PM	#63
GW_OK Senior Member Location: Oklahoma Join Date: Sep 2009 Posts: 411	Huh, must have missed that.

05-29-2011, 11:28 AM	#64
james hadfield Moderator Cambridge, UK Community Forum Location: Cambridge, UK Join Date: Feb 2008 Posts: 221	There is also a PowerPoint presentation in PDF format on the Illumina site which gives more infomration on the poster. One interesting thing is the error plots for all runs they show. Something I have not noticed before (need to look next week) is that all the plots show a kind of step-like profile increase in quality over the first 20 or so bases. Then there is a drop off in quality as run length increases. However this is not a simple drop as shown on slide 14. Slide 18 shows HiSeq v MiSeq for 100bp runs of an E. coli library. Both show a drop in quality at about which recovers immediatley after, however it is at 75/65bp for Hi/MiSeq respectively. This may be an error in the library??? Slide 5 shows some lovely data on amplicon resequencing and mutation detection. It makes me wonder what the final reports are going to look like when this gets into a non-bioinformaticians hands?

07-09-2011, 07:22 AM	#65
raonyguimaraes Member Location: Belo Horizonte - Brazil Join Date: Jun 2010 Posts: 38	Just found this hilarious videos about MYSEQ and HISEQ http://www.youtube.com/watch?v=GUr17pHezUo http://www.youtube.com/watch?v=_xBwWMT7PgA http://www.youtube.com/watch?v=gStCvyGpnRU http://www.youtube.com/watch?v=G20F7GkHsmY

07-21-2011, 02:13 AM	#67
razibus Member Location: France Join Date: May 2011 Posts: 25	I think this service will be provided via GoldenGate platform.

07-22-2011, 07:51 AM	#69
krobison Senior Member Location: Boston area Join Date: Nov 2007 Posts: 747	Given that GoldenGate is a technology upstream of their arrays, could you expand on how this is going to be applied to sequencing?

07-22-2011, 08:10 AM	#70
james hadfield Moderator Cambridge, UK Community Forum Location: Cambridge, UK Join Date: Feb 2008 Posts: 221	The Illumina GoldenGate PCR is used for lower plex genotyping and other applications on arrays. The PCR is likely to be modified such that a set of locus specific primers have regions with complimentarity to Illumina adapters. GoldenGate on arrays allows up to 3072 multiplex reactions. This offers the possibility of 96-384 and even up to 3072 loci (e.g. exons) being targeted by PCR in a standard 96 or 384 well plate from very low input DNA. And generating sequence ready amplicons which would fit neatly into a MiSeq PE run. The whole workflow including PCR could be completed in one day with results returned, analysed the next morning depending on run configuration. This workflow would compete with other targeting approaches aimed at low multiplexing and not with whole exome resequencing for instance. As it could be 96 or 384 well based I could see a panel of cancer genes being resequenced at every exon as a standard product any user could buy from Illumina, add their samples to and get results on really quickly. In fact I've asked to do exactly that. The GoldenGate product was announced as being launched at the same time as MiSeq in September/October.

07-22-2011, 08:13 AM	#71
james hadfield Moderator Cambridge, UK Community Forum Location: Cambridge, UK Join Date: Feb 2008 Posts: 221	The Illumina GoldenGate PCR is used for lower plex genotyping and other applications on arrays. The PCR is likely to be modified such that a set of locus specific primers have regions with complimentarity to Illumina adapters. GoldenGate on arrays allows up to 3072 multiplex reactions. This offers the possibility of 96-384 and even up to 3072 loci (e.g. exons) being targeted by PCR in a standard 96 or 384 well plate from very low input DNA. And generating sequence ready amplicons which would fit neatly into a MiSeq PE run. The whole workflow including PCR could be completed in one day with results returned, analysed the next morning depending on run configuration. This workflow would compete with other targeting approaches aimed at low multiplexing and not with whole exome resequencing for instance. As it could be 96 or 384 well based I could see a panel of cancer genes being resequenced at every exon as a standard product any user could buy from Illumina, add their samples to and get results on really quickly. In fact I've asked to do exactly that. The GoldenGate product was announced as being launched at the same time as MiSeq in September/October.

07-22-2011, 08:17 AM	#72
james hadfield Moderator Cambridge, UK Community Forum Location: Cambridge, UK Join Date: Feb 2008 Posts: 221	GW_OK here is an answer from my measurements. A MiSeq flowcell is 1.5x23mm see a picture here… Core-Genomics blog, however only a portion of this is imaged as far as I am aware.

08-31-2011, 06:13 AM	#75
krobison Senior Member Location: Boston area Join Date: Nov 2007 Posts: 747	In addition to Illumina's analysis, there are analyses from Ion (referenced in my blog item) and from Edge Bio (which provides Ion as a service).