TruSeq Sample Prep, Enrichment PCR primer sequences

EdibleMetal · 02-18-2011, 02:00 PM

Has anybody made enrichment PCR primers for Illumina's new TruSeq DNA Sample Prep kit that work? If so, would you share the primer sequences with me? Thanks!

BTS · 02-23-2011, 08:40 AM

Are you looking to index samples? TruSeq has the PCR primer sequences available. Maybe this is along the lines of your question, but does anyone know why the primers are so long and include the indexes (thus needing several different primers in the cocktail)? Wouldn't it be more practical to put the enrichment PCR primers just outside of the indexes so two primers could be used for any index sequence? Maybe I'm missing something here? Thanks!

elaney_k · 02-23-2011, 09:11 AM

Quote:

Originally Posted by BTS

Maybe this is along the lines of your question, but does anyone know why the primers are so long and include the indexes (thus needing several different primers in the cocktail)?

Hi BTS, the sequences I think you're referring to that are available from Illumina are the new longer indexed adapters used in the TruSeq kits as opposed to the multiplex PCR primers used previously. There is only one set of enrichment primers that are just outside of the index.

Elaine

alisrpp · 02-23-2011, 09:19 AM

If you want to do a multiplexed library you must need the indexes. At the beginning Illumina was using the indexes as a 3rd primer, so at the step of the enrichment PCR you added the 2 PCR primers + the index. But that way to proceed was not working well so the changed the protocol and added the index to the 2.0 PCR primer getting a long primer.
But I have a new question. If you have the old PCR primers, I mean, the PCR primers separately from the indexes, can you do an annealing of the PCR primer 2.0 with the index and get a long indexed PCR primer 2.0? Did anyone try that?
Thanks.

EdibleMetal · 02-23-2011, 09:28 AM

The old kits used to have you ligate on the adapter and then add extra bases to that adapter using enrichment PCR primers with a 5' overhang (which could include a barcode if you so choose). The new TruSeq DNA sample prep kit has the barcode embedded in the sequence of the adapter. With this kit, it does not seem as if enrichment PCR primers add any extra sequence to this adapter to make it flowcell compatible, so my question is still: has anybody made enrichment PCR primers for Illumina's new TruSeq DNA Sample Prep kit that work?

BTS · 02-23-2011, 11:51 AM

Thanks for clarifying that Elaine. I was confused when reading through the document Illumina released and called tech support about it. Definitely makes more sense. We will probably be trying to design our own PCR primers and testing them which would get at EdibleMetal's question. I will try to remember to report when we get that far!

EdibleMetal · 02-23-2011, 05:57 PM

Yes BTS! If you do design primers and test them out, I would really appreciate it if you let me know how it works out for you. I also plan to design primers and test them out, so I'll let you know how it works out on my end.

Illumina did not release the sequences of the enrichment PCR primers in the new TruSeq DNA library prep kits, so I cannot tell if the primers provided in the kit actually add any extra nucleotides (i.e. 5' overhang) that are crucial for primers to anneal during bridge amplification. I also can't seem to find the sequences for the sequencing primers (H6, H7, H8) provided with Version 2 of the cBot kit. So when I go to design the primers, I just want to make sure that all of these aforementioned primer-annealing sites are embedded in the new TruSeq adapter sequence. I'll stop babbling now. Thanks for listening.

james hadfield · 02-24-2011, 01:20 AM

The capture primers will do just that, capture library fragments that are complementary. Thye probably have a biotin on for pulling out during the prep. But all the seqeucne needed for bridge amplification and seqeucing is in the TruSeq adpater sequence.

Illumina TechSupport can supply these sequences if you ask them. However the actual sequence primer used is not published, I believe it is the same as the current one.

EdibleMetal · 03-09-2011, 04:02 PM

Hi James,

Illumina TechSupport will not provide these enPCR sequences to me. Perhaps they will provide these sequences to you?

RNAseqer · 03-09-2011, 08:41 PM

Looks like Bioo has 48 DNA sequencing adapters with barcodes embedded in the adapter sequence. They provided me with their sequence information when I called. Their enrichment primers are also included in their adapter barcoding kits which are reasonably priced $390.

epistatic · 04-12-2011, 10:28 PM

Be careful with the Bioo 48-sample kit. I ordered it and luckily hadn't used it yet when they informed me that two adapters in the 48 had sequences of other adapters and would need to be replaced. The "redundant adapters" were 32 and 33.

I'm now less confident of the kit and manufacturer, but at least they caught the mistake. I wonder if anyone had to sequence to find this out...

Bioo Scientific · 04-13-2011, 12:15 PM

Hi Epistatic,

We are sorry about the problem you experienced. We’ve fixed this issue and everyone who has received the 48 barcode kit has the proper 32 and 33 adapters. This only affected kits that were shipped kits during the last week of March.

We take quality seriously and guarantee all our sequencing products. Let us know if you have any questions.

Dawn
Bioo Scientific

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02-18-2011, 02:00 PM	#1
EdibleMetal Junior Member Location: US Join Date: Jul 2010 Posts: 6	TruSeq Sample Prep, Enrichment PCR primer sequences Has anybody made enrichment PCR primers for Illumina's new TruSeq DNA Sample Prep kit that work? If so, would you share the primer sequences with me? Thanks!

03-09-2011, 08:41 PM	#10
RNAseqer Member Location: London Join Date: Sep 2010 Posts: 22	barcoded adapters Looks like Bioo has 48 DNA sequencing adapters with barcodes embedded in the adapter sequence. They provided me with their sequence information when I called. Their enrichment primers are also included in their adapter barcoding kits which are reasonably priced $390.

02-23-2011, 08:40 AM	#2
BTS Member Location: Wisconsin Join Date: Sep 2010 Posts: 19	Are you looking to index samples? TruSeq has the PCR primer sequences available. Maybe this is along the lines of your question, but does anyone know why the primers are so long and include the indexes (thus needing several different primers in the cocktail)? Wouldn't it be more practical to put the enrichment PCR primers just outside of the indexes so two primers could be used for any index sequence? Maybe I'm missing something here? Thanks!

02-23-2011, 09:19 AM	#4
alisrpp Member Location: New York Join Date: Dec 2010 Posts: 40	If you want to do a multiplexed library you must need the indexes. At the beginning Illumina was using the indexes as a 3rd primer, so at the step of the enrichment PCR you added the 2 PCR primers + the index. But that way to proceed was not working well so the changed the protocol and added the index to the 2.0 PCR primer getting a long primer. But I have a new question. If you have the old PCR primers, I mean, the PCR primers separately from the indexes, can you do an annealing of the PCR primer 2.0 with the index and get a long indexed PCR primer 2.0? Did anyone try that? Thanks.

02-23-2011, 09:28 AM	#5
EdibleMetal Junior Member Location: US Join Date: Jul 2010 Posts: 6	The old kits used to have you ligate on the adapter and then add extra bases to that adapter using enrichment PCR primers with a 5' overhang (which could include a barcode if you so choose). The new TruSeq DNA sample prep kit has the barcode embedded in the sequence of the adapter. With this kit, it does not seem as if enrichment PCR primers add any extra sequence to this adapter to make it flowcell compatible, so my question is still: has anybody made enrichment PCR primers for Illumina's new TruSeq DNA Sample Prep kit that work?

02-23-2011, 11:51 AM	#6
BTS Member Location: Wisconsin Join Date: Sep 2010 Posts: 19	Thanks for clarifying that Elaine. I was confused when reading through the document Illumina released and called tech support about it. Definitely makes more sense. We will probably be trying to design our own PCR primers and testing them which would get at EdibleMetal's question. I will try to remember to report when we get that far!

02-23-2011, 05:57 PM	#7
EdibleMetal Junior Member Location: US Join Date: Jul 2010 Posts: 6	Yes BTS! If you do design primers and test them out, I would really appreciate it if you let me know how it works out for you. I also plan to design primers and test them out, so I'll let you know how it works out on my end. Illumina did not release the sequences of the enrichment PCR primers in the new TruSeq DNA library prep kits, so I cannot tell if the primers provided in the kit actually add any extra nucleotides (i.e. 5' overhang) that are crucial for primers to anneal during bridge amplification. I also can't seem to find the sequences for the sequencing primers (H6, H7, H8) provided with Version 2 of the cBot kit. So when I go to design the primers, I just want to make sure that all of these aforementioned primer-annealing sites are embedded in the new TruSeq adapter sequence. I'll stop babbling now. Thanks for listening.

02-24-2011, 01:20 AM	#8
james hadfield Moderator Cambridge, UK Community Forum Location: Cambridge, UK Join Date: Feb 2008 Posts: 221	The capture primers will do just that, capture library fragments that are complementary. Thye probably have a biotin on for pulling out during the prep. But all the seqeucne needed for bridge amplification and seqeucing is in the TruSeq adpater sequence. Illumina TechSupport can supply these sequences if you ask them. However the actual sequence primer used is not published, I believe it is the same as the current one.

03-09-2011, 04:02 PM	#9
EdibleMetal Junior Member Location: US Join Date: Jul 2010 Posts: 6	Hi James, Illumina TechSupport will not provide these enPCR sequences to me. Perhaps they will provide these sequences to you?

04-12-2011, 10:28 PM	#11
epistatic Senior Member Location: Dronning Maud Land Join Date: Mar 2009 Posts: 129	Be careful with the Bioo 48-sample kit. I ordered it and luckily hadn't used it yet when they informed me that two adapters in the 48 had sequences of other adapters and would need to be replaced. The "redundant adapters" were 32 and 33. I'm now less confident of the kit and manufacturer, but at least they caught the mistake. I wonder if anyone had to sequence to find this out...

04-13-2011, 12:15 PM	#12
Bioo Scientific Registered Vendor Location: Austin, Tx Join Date: Oct 2009 Posts: 99	Hi Epistatic, We are sorry about the problem you experienced. We’ve fixed this issue and everyone who has received the 48 barcode kit has the proper 32 and 33 adapters. This only affected kits that were shipped kits during the last week of March. We take quality seriously and guarantee all our sequencing products. Let us know if you have any questions. Dawn Bioo Scientific