Nimblegen arrays for targeted genome enrichment - Page 3

lambroso · 08-12-2008, 07:46 AM

Hi Sigusn,
I did the elution similiar to your method. I placed the bottom piece of a Corning hybridization chamber on a piece of tin foil in a 100 degree heating block, with out the blocks in it. Then put my slide on it and added 2mls of heated water. I know 2mls sounds like alot, but I found that it kept the slide covered for the total elution time and could be gently mixed without spilling off. I used the hyb chamber in case anything spilled off the slide. Lucky for us we have a good speed vac. But, the bottom line is I still haven't gotten this to work yet as verified by running the speed vaced elution on a gel. I started out with 25ug and if I had similiar results as Okou, who recovered .7-1.2ug, I should have been able to see it on a gel. How long are you eluting for? I'm not sure what to try next. Are you hybridizing for 60 hours? I was just doing it over night but may need to increase it. Also curious about what Nimblegen's special unit for elution is like.
Thanks again for you thought on this.

sigusn · 08-12-2008, 10:12 AM

Hi
I hybridized for 65h. After elution I used speedvac to dry down the sample, then dissolved in water. I did not get as much as Okou et al but enough to perform the PCR step and then the PCR product was seen on gel and on bioanalyzer. This was true for 4 out of 6 samples i worked with, I have not found out what happened to the two that gave no PCR product. Probably low quality of the input DNA.
I would guess that the elution product is single stranded DNA (the Nimblegen arrays are only designed on the forward strand) and single stranded DNA is not seen on agarose gel with EtBr staining.

fgibson · 08-22-2008, 05:11 AM

has anyone tried using pools of genomic DNA on the nimblegen sequence capture arrays?

sigusn · 08-26-2008, 01:22 AM

Yes, I have tried pooling 4 samples and use the Nimblegen Seqcap arrays. With average sequence coverage of 40-50X I think I can say that works OK.

rosatoc · 08-28-2008, 08:59 AM

My understanding is that a custom seq cap array from NimbleGen runs around $1K. As we prepare to offer a seq cap in this core facility I'm glad to know there is a thread I can come to for tips on technical points. Is there a reference for the PCR amplification post elution (Okou et al?)? Since the initial expectation for sequencing seq cap material was for the 454, I'm pleased to know that seq cap is working with the Illumina (the one platform we have). Thanks!

sigusn · 08-31-2008, 12:08 PM

Some references>
Okou et al. Nat Methods. 2007 Nov;4(11):907-9
Excelent method description in the supplement

Hodges et al. Nat Genet. 2007 Dec;39(12):1522-7.
Describe the sequence capture followed by Illumina Genome analyzer

The custom made sequence capture arrays from Nimblegen now come with full protocol for the sample prep, should work for Illumina if you just use the right oligos.

dvh · 09-06-2008, 03:08 PM

Edited 10 SEPT 2008: I wish to withdraw this post - see post 48 below:

Hi,
We've just run our first nimblegen seq cap service (i.e. they did the probe design/hyb/elute/pcr amp steps) sample on a solexa flowcell. We confirmed the returned sample looked OK on bionanalyser.
No problems with solexa library prep.
Flowcell worked well (GAII) for other libraries.
After removing the 454 adapters, almost no reads map to the regions we asked for on the chip (custom design 1.5Mb). However the reads map very well to the human genome (maq alignment to whole genome, >90% map, error rate 1.0%). But to a few distinct regions which look like they might be repeat regions.

We have some other samples to run, and will repeat the library prep, but am a bit concerned by this. Wondered if anyone else has had any difficulties?

david

dvh · 09-10-2008, 12:37 PM

I wish to withdraw the post (#47) above.

There was a sample mix up on the flowcell. Lane 1 was actually lane 8 etc. Found this out from the PhiX control, the fact we had mixed sample types, and careful inspection of the data. Worth pointing out to the list that it is VERY easy to flip the 8-well tube of samples (or perhaps flowcell) in the cluster station.

To apologise for the earlier post, here are some results from the first lane we've looked at. Single end solexa reads. We have only very crudely dealt with the (unwanted) adapter sequence used by nimblegen for PCR amplification at present, much more optimisation is possible which will improve both number of reads and quality of reads aligning. Using maq map -n 3 to the whole genome, 1.3m reads map with MQ 70-99 i.e. uniquely with high confidence. Of these 85% map to the regions we selected for the seq cap chip. So it works. Somewhat variable coverage, I'll try and post some stats once we've done some more analysis.

david

poliveira · 09-19-2008, 07:26 AM

Hi all!

My first time here so be gentle

My question is: have you any idea whether the sequencing results will allow the user to distinguish strand orientation... This because I am interested in transcriptome analysis and the strand info is crutial... Let me know please, I'm drowning in papers...
Cheers to all and thanks to the threading you all are introducing me to a brand new world!
ciao!

bioinfosm · 09-19-2008, 10:48 AM

Not sure if I understood you right, but Yes. The alignment algorithm will tell you if the sequence aligned to forward/reverse strand of the reference sequence you provide..

Chipper · 09-19-2008, 12:17 PM

Quote:

Originally Posted by poliveira

Hi all!

My first time here so be gentle

My question is: have you any idea whether the sequencing results will allow the user to distinguish strand orientation... This because I am interested in transcriptome analysis and the strand info is crutial... Let me know please, I'm drowning in papers...
Cheers to all and thanks to the threading you all are introducing me to a brand new world!
ciao!

You will only be able to tell the direction / strand of the sequence you read, not the transcript. If you have polymorphisms in your exons you might see some strand specificity though. But for transcriptiome sequencing you do not need the enrichment arrays I guess.

ATGC_man · 01-08-2009, 12:23 AM

Hello,

HD2 is available from Nimblegen !

Anyone have more information about that ?

sci_guy · 01-08-2009, 01:58 PM

Nimblegen are hosting a webinar very soon. See here.

vasvale · 01-27-2009, 09:23 AM

imp[rovements for the library prep in this paper:
Nat Methods. 2008 Dec;5(12):1005-10.

A large genome center's improvements to the Illumina sequencing system.

Quail MA, Kozarewa I, Smith F, Scally A, Stephens PJ, Durbin R, Swerdlow H,
Turner DJ.

Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton,
Cambridgeshire, CB10 1SA, UK.

giakr84 · 05-12-2010, 08:04 AM

Hi all, do you know an alternative method to perform a microarray experiment using 454 Junior? (no Nimblegen)
Thanks

tanmoy_img · 10-26-2010, 06:54 AM

Hi.....
I want some information regarding nimbulgen sequence capture of mouse genome.I want to sequence4.5 Mbp region on x chromosome of few mouse samples.can anybody share there experience with mouse samples.All kind of recommendations are welcome.Please send me some references or protocols or articles on the same.Please help.
Best regards,
Tanmoy

sigusn · 10-26-2010, 08:14 AM

Hi
We have done Nimblegen sequence capture on dogs, horses, cows, mouse, humans, etc. All works fine. Just using the standard protocol from Nimblegen+Illumina should work fine, or use your own library preparation variants.

tanmoy_img · 10-26-2010, 12:08 PM

Hi sigusn....
Thank you for your reply....can u suggest me some research papers.
rgds,
T

sigusn · 10-26-2010, 12:54 PM

We don't have anything published on Mouse yet. But here are some papers:
This one tests Nimblegen capture
http://www.ncbi.nlm.nih.gov/pubmed/20562348
Using Nimblegen capture on Horses
http://www.ncbi.nlm.nih.gov/pubmed/19892987
On cattle
PMID: 20865119
Nimblgen has a collection of papers, user guides and protocols
http://www.nimblegen.com/products/lit/index.html
I hope that helps something
regards,
Snaevar

tanmoy_img · 10-27-2010, 12:57 AM

thank you very much for the help.
regards,
Tanmoy

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08-22-2008, 05:11 AM	#43
fgibson Junior Member Location: Imperial Join Date: Jun 2008 Posts: 1	DNA pooling with nimblegen sequence capture arrays has anyone tried using pools of genomic DNA on the nimblegen sequence capture arrays?

08-26-2008, 01:22 AM	#44
sigusn Member Location: Cambridge, MA Join Date: Aug 2008 Posts: 19	pooled DNA Yes, I have tried pooling 4 samples and use the Nimblegen Seqcap arrays. With average sequence coverage of 40-50X I think I can say that works OK.

09-10-2008, 12:37 PM	#48
dvh Member Location: london, uk Join Date: Jul 2008 Posts: 35	first experience with nimblegen seq cap on GAII I wish to withdraw the post (#47) above. There was a sample mix up on the flowcell. Lane 1 was actually lane 8 etc. Found this out from the PhiX control, the fact we had mixed sample types, and careful inspection of the data. Worth pointing out to the list that it is VERY easy to flip the 8-well tube of samples (or perhaps flowcell) in the cluster station. To apologise for the earlier post, here are some results from the first lane we've looked at. Single end solexa reads. We have only very crudely dealt with the (unwanted) adapter sequence used by nimblegen for PCR amplification at present, much more optimisation is possible which will improve both number of reads and quality of reads aligning. Using maq map -n 3 to the whole genome, 1.3m reads map with MQ 70-99 i.e. uniquely with high confidence. Of these 85% map to the regions we selected for the seq cap chip. So it works. Somewhat variable coverage, I'll try and post some stats once we've done some more analysis. david

10-26-2010, 06:54 AM	#56
tanmoy_img Junior Member Location: Czech republic Join Date: Oct 2010 Posts: 3	An urgent help needed regarding sequence caputure Hi..... I want some information regarding nimbulgen sequence capture of mouse genome.I want to sequence4.5 Mbp region on x chromosome of few mouse samples.can anybody share there experience with mouse samples.All kind of recommendations are welcome.Please send me some references or protocols or articles on the same.Please help. Best regards, Tanmoy

08-12-2008, 07:46 AM	#41
lambroso Junior Member Location: us Join Date: Jul 2008 Posts: 2	Hi Sigusn, I did the elution similiar to your method. I placed the bottom piece of a Corning hybridization chamber on a piece of tin foil in a 100 degree heating block, with out the blocks in it. Then put my slide on it and added 2mls of heated water. I know 2mls sounds like alot, but I found that it kept the slide covered for the total elution time and could be gently mixed without spilling off. I used the hyb chamber in case anything spilled off the slide. Lucky for us we have a good speed vac. But, the bottom line is I still haven't gotten this to work yet as verified by running the speed vaced elution on a gel. I started out with 25ug and if I had similiar results as Okou, who recovered .7-1.2ug, I should have been able to see it on a gel. How long are you eluting for? I'm not sure what to try next. Are you hybridizing for 60 hours? I was just doing it over night but may need to increase it. Also curious about what Nimblegen's special unit for elution is like. Thanks again for you thought on this.

08-12-2008, 10:12 AM	#42
sigusn Member Location: Cambridge, MA Join Date: Aug 2008 Posts: 19	Hi I hybridized for 65h. After elution I used speedvac to dry down the sample, then dissolved in water. I did not get as much as Okou et al but enough to perform the PCR step and then the PCR product was seen on gel and on bioanalyzer. This was true for 4 out of 6 samples i worked with, I have not found out what happened to the two that gave no PCR product. Probably low quality of the input DNA. I would guess that the elution product is single stranded DNA (the Nimblegen arrays are only designed on the forward strand) and single stranded DNA is not seen on agarose gel with EtBr staining.

08-28-2008, 08:59 AM	#45
rosatoc Member Location: Oregon Join Date: Apr 2008 Posts: 22	My understanding is that a custom seq cap array from NimbleGen runs around $1K. As we prepare to offer a seq cap in this core facility I'm glad to know there is a thread I can come to for tips on technical points. Is there a reference for the PCR amplification post elution (Okou et al?)? Since the initial expectation for sequencing seq cap material was for the 454, I'm pleased to know that seq cap is working with the Illumina (the one platform we have). Thanks!

08-31-2008, 12:08 PM	#46
sigusn Member Location: Cambridge, MA Join Date: Aug 2008 Posts: 19	Some references> Okou et al. Nat Methods. 2007 Nov;4(11):907-9 Excelent method description in the supplement Hodges et al. Nat Genet. 2007 Dec;39(12):1522-7. Describe the sequence capture followed by Illumina Genome analyzer The custom made sequence capture arrays from Nimblegen now come with full protocol for the sample prep, should work for Illumina if you just use the right oligos.

09-06-2008, 03:08 PM	#47
dvh Member Location: london, uk Join Date: Jul 2008 Posts: 35	first experience with nimblegen seq cap on GAII - ?problem Edited 10 SEPT 2008: I wish to withdraw this post - see post 48 below: Hi, We've just run our first nimblegen seq cap service (i.e. they did the probe design/hyb/elute/pcr amp steps) sample on a solexa flowcell. We confirmed the returned sample looked OK on bionanalyser. No problems with solexa library prep. Flowcell worked well (GAII) for other libraries. After removing the 454 adapters, almost no reads map to the regions we asked for on the chip (custom design 1.5Mb). However the reads map very well to the human genome (maq alignment to whole genome, >90% map, error rate 1.0%). But to a few distinct regions which look like they might be repeat regions. We have some other samples to run, and will repeat the library prep, but am a bit concerned by this. Wondered if anyone else has had any difficulties? david Last edited by dvh; 09-10-2008 at 12:46 PM.

09-19-2008, 07:26 AM	#49
poliveira Junior Member Location: Portugal Join Date: Sep 2008 Posts: 1	Hi all! My first time here so be gentle My question is: have you any idea whether the sequencing results will allow the user to distinguish strand orientation... This because I am interested in transcriptome analysis and the strand info is crutial... Let me know please, I'm drowning in papers... Cheers to all and thanks to the threading you all are introducing me to a brand new world! ciao!

09-19-2008, 10:48 AM	#50
bioinfosm Senior Member Location: USA Join Date: Jan 2008 Posts: 482	Not sure if I understood you right, but Yes. The alignment algorithm will tell you if the sequence aligned to forward/reverse strand of the reference sequence you provide..

01-08-2009, 12:23 AM	#52
ATGC_man Member Location: france Join Date: Nov 2008 Posts: 10	Hello, HD2 is available from Nimblegen ! Anyone have more information about that ?

01-08-2009, 01:58 PM	#53
sci_guy Member Location: Sydney, Australia Join Date: Jan 2008 Posts: 83	Nimblegen are hosting a webinar very soon. See here.

01-27-2009, 09:23 AM	#54
vasvale Member Location: Seattle Join Date: Mar 2008 Posts: 29	imp[rovements for the library prep in this paper: Nat Methods. 2008 Dec;5(12):1005-10. A large genome center's improvements to the Illumina sequencing system. Quail MA, Kozarewa I, Smith F, Scally A, Stephens PJ, Durbin R, Swerdlow H, Turner DJ. Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridgeshire, CB10 1SA, UK.

05-12-2010, 08:04 AM	#55
giakr84 Junior Member Location: italy Join Date: Mar 2010 Posts: 2	Hi all, do you know an alternative method to perform a microarray experiment using 454 Junior? (no Nimblegen) Thanks

10-26-2010, 08:14 AM	#57
sigusn Member Location: Cambridge, MA Join Date: Aug 2008 Posts: 19	Hi We have done Nimblegen sequence capture on dogs, horses, cows, mouse, humans, etc. All works fine. Just using the standard protocol from Nimblegen+Illumina should work fine, or use your own library preparation variants.

10-26-2010, 12:08 PM	#58
tanmoy_img Junior Member Location: Czech republic Join Date: Oct 2010 Posts: 3	Hi sigusn.... Thank you for your reply....can u suggest me some research papers. rgds, T

10-26-2010, 12:54 PM	#59
sigusn Member Location: Cambridge, MA Join Date: Aug 2008 Posts: 19	We don't have anything published on Mouse yet. But here are some papers: This one tests Nimblegen capture http://www.ncbi.nlm.nih.gov/pubmed/20562348 Using Nimblegen capture on Horses http://www.ncbi.nlm.nih.gov/pubmed/19892987 On cattle PMID: 20865119 Nimblgen has a collection of papers, user guides and protocols http://www.nimblegen.com/products/lit/index.html I hope that helps something regards, Snaevar

10-27-2010, 12:57 AM	#60
tanmoy_img Junior Member Location: Czech republic Join Date: Oct 2010 Posts: 3	thank you very much for the help. regards, Tanmoy