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Old 02-21-2012, 03:21 AM   #1
Vinz
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Default Dual indexing construct for MiSeq - where to put the barcode for index2?

We would like to use the second indexing read on the MiSeq for amplicon sequencing (not using the Illumina Custom Amplicon Sequencing). Has anyone successfully designed the adaptors containing index2? Where would you put the barcodes?
I assume "TrueSeq Universal Adapter" from the "Illumina-Customer-Sequence-Letter" would be the sequence to start.
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Old 02-22-2012, 03:31 PM   #2
koadman
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Hi Vinz, I looked into this and the information that Illumina distributes to customers does not contain the full length adapter sequences as far as I know. You might also be interested in this thread where csquared helpfully suggested that the 2nd barcode is inside the other adapter and is primed directly from the flowcell annealing sequence. Reading Illumina's instrument operation manuals appears to confirm this, they suggest there are some chemistry-only dark cycles prior to reading the other index in their standard protocol and these presumably synthesize the bases between the flow cell annealing region and the index region.

However, if you are doing amplicon sequencing you probably have the flexibility to design your own sequencing primers in which case you might be able to follow a design like what Kircher et al have described. That would have the advantage of not wasting 9 cycles of reagents.
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Old 02-24-2012, 02:55 AM   #3
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Thanks for the link to the paper of Kircher et al. That is indeed helpful.
The way the 2nd barcode is sequenced is nicely described in the Dual Indexing Training for all that have access. It just does not show any sequences.
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Old 02-24-2012, 07:42 AM   #4
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The video in that training is the best explanation I've seen yet of the dual index adapter structure and sequencing process. Interesting that the 2nd index is sequenced while the template molecule is bridged. Thanks Vinz!
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Old 05-08-2012, 08:08 AM   #5
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Hey guys. I'm in the process of making my own Truseq-esque double indexing adapters for MiSeq, and (as you've seen) Illumina will not be forthcoming with the sequences...so I had to figure it out for myself and will share it with you.

I just sequenced a standard single-indexed TruSeq library with the Nextera/Amplicon protocol and the below describes my observations.
It may not help with figuring out the nextera sequences but probably contains some information that will help.

I'm just about to run libraries with my own homebrew double indexing design, will post when/if it turns out well.

Attached Files
File Type: pdf Double Indexing with Standard MiSeq Recipe.pdf (46.8 KB, 745 views)
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Old 05-08-2012, 08:10 AM   #6
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Oh and page 5 of this document has as nice diagram of the process, so far the chemistry has seemed the same on the MiSeq.
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File Type: pdf SequencingNexteraLibraries_onHiSeq_15032071_A.pdf (413.9 KB, 841 views)
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Old 05-10-2012, 10:47 PM   #7
Vinz
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Meanwhile we figured out how to do the dual indexing.
What you have colored green are the 7 bases that are cut with an restriction enzyme and later added again by the 7 dark cycles. We hypothesize the restriction enzyme is DpnI. Recognition site GATC, cutting after GA.
This green sequence should be as it is on the left side of the index. After the index the ACAC is repeated as this matches the 5' end of read 1 sequencing primer and then you go on with the red part...
This works very well for us on the MiSeq for dual indexing with the primers of the cartridge.
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Old 06-14-2012, 11:48 AM   #8
gnomers
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Quote:
Originally Posted by ECO View Post
I'm just about to run libraries with my own homebrew double indexing design, will post when/if it turns out well.
By "homebrew," do you mean that you were using custom Read 1, i7 index and Read 2 primers? I am contemplating this strategy for dual indexed amplicons in order to avoid sequencing the constant regions that anneal to the PCR template.

Either way, how did it turn out?
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Old 06-19-2012, 07:22 AM   #9
seq_yak
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Default MiSeq Dual-indexing

Quote:
Originally Posted by Vinz View Post
Meanwhile we figured out how to do the dual indexing.
What you have colored green are the 7 bases that are cut with an restriction enzyme and later added again by the 7 dark cycles. We hypothesize the restriction enzyme is DpnI. Recognition site GATC, cutting after GA.
This green sequence should be as it is on the left side of the index. After the index the ACAC is repeated as this matches the 5' end of read 1 sequencing primer and then you go on with the red part...
This works very well for us on the MiSeq for dual indexing with the primers of the cartridge.
I have a library design based on Kirchner et al. as well and I'm planning to run it on the MiSeq. So, did you have to tweak the sample sheet of the MiSeq or did you use the standard Nextera program? Maybe you could even share your sample sheet here?

Thanks,
seq_yak

Last edited by seq_yak; 06-19-2012 at 07:24 AM. Reason: a lot of typos
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Old 06-21-2012, 04:56 AM   #10
Vinz
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I have attached a recent sample sheet example.
The sequence of index1 (I7) needs to be reverse complemented of what you ordered as primer. The sequence of index2 is just as the primer was synthesized.
However, this will use the index and read primers of the cartridge. If you want to add your own primers you need to add this information.
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File Type: txt sampleSheet_example.txt (2.5 KB, 482 views)
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Old 06-21-2012, 05:30 AM   #11
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Does that mean that using the following primers you could create your own dual indexed amplicon library?

AATGATACGGCGACCACCGAGA{TCTACAC}[i5 index][custom fwd]
CAAGCAGAAGACGGCATACGAGAT[i7 index][custom rev]

Dark cycles in curly brackets{ }
Read 1 with custom fwd
Index i7 with reverse compliment of custom rev
Index i5 same as Illumina's
Read 2 with custom rev

We'd be very interested in this for potential 16s studies as could then feasibly multiplex 96, or even 384 samples in one MiSeq run.
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Old 06-21-2012, 05:41 AM   #12
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I think this should do it.
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Old 06-21-2012, 06:58 AM   #13
seq_yak
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Quote:
Originally Posted by Vinz View Post
I have attached a recent sample sheet example.
The sequence of index1 (I7) needs to be reverse complemented of what you ordered as primer. The sequence of index2 is just as the primer was synthesized.
However, this will use the index and read primers of the cartridge. If you want to add your own primers you need to add this information.
Hi Vinz,

thanks for the input. So, if I understood you right you're able to use the primers of the cartridge and the sample sheet you posted to sequence such a TruSeq-esque library (attached)?
Attached Files
File Type: pdf double_index_library.pdf (10.2 KB, 890 views)
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Old 06-21-2012, 07:11 AM   #14
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Hi seq_yak,
yes, with the design you attached things should work well. You do not need to add additional primers and with the sample sheet MiSeq should give you nice sequences. Good luck!
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Old 06-25-2012, 03:25 AM   #15
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Quote:
Originally Posted by Vinz View Post
Meanwhile we figured out how to do the dual indexing.
What you have colored green are the 7 bases that are cut with an restriction enzyme and later added again by the 7 dark cycles. We hypothesize the restriction enzyme is DpnI. Recognition site GATC, cutting after GA.
This green sequence should be as it is on the left side of the index. After the index the ACAC is repeated as this matches the 5' end of read 1 sequencing primer and then you go on with the red part...
So that would be a hemi-methylated DpnI site? With the flowcell oligo contributing the methylated adenine required for DpnI to cut?

--
Phillip
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Old 06-27-2012, 09:54 AM   #16
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Ok sorry for the delay all...please see attached document for my results/experience. There is a review of standard truseq, the design that I came up with with help from this thread, and an example samplesheet.

Everything comes with my standard disclaimer of "if you don't know what you're doing you WILL mess something up" and...this is based on one successful run of testing...so this is very experimental.
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Old 06-28-2012, 07:26 AM   #17
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Quote:
Originally Posted by pmiguel View Post
So that would be a hemi-methylated DpnI site? With the flowcell oligo contributing the methylated adenine required for DpnI to cut?

--
Phillip
Hi Philip,
that was the idea. But ECO pointed out that it is probably uracil at some position and some uracil dependant cleavage.
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Old 06-29-2012, 07:30 AM   #18
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I could definitely be wrong though!! It seems crazy to rely on restriction when more specificity would be achieved by uracil dependent cleavage.

The flow cell primer having a uracil at that position is a bit of a clue too...assuming that info is correct.
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Old 07-03-2012, 08:46 AM   #19
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Hi all, I'm new to NGS so approach this post with a healthy dose of skepticism.

I'm in the process of designing PCR primers for a targeted approach to sequencing dual indexed amplicons on the MiSeq. After reading ECO's posts, this is what I've deduced about primer design:

FORWARD PCR PRIMER:
5 AATGATACGGCGACCACCGAGA{TCTACAC}nnnnnnnnACACTCTTTCCCTACACGACGCTCTTCCGATCT[Forward primer sequence]
REVERSE PCR PRIMER:
5 CAAGCAGAAGACGGCATACGAGATnnnnnnnnGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT[Reverse primer sequence]

...where bold = i5 and i7 indices, respectively.

This confuses me because the sequences following the indices are different from those introduced by the transposon. According to Illumina's Customer Sequence Letter, a targeted PCR strategy that mimics samples prepped using Nextera would have primers that appear as follows:

FORWARD PCR PRIMER:
5' - AATGATACGGCGACCACCGAGA{TCTACAC}nnnnnnnnTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG[Forward primer sequence]
REVERSE PCR PRIMER:
5 CAAGCAGAAGACGGCATACGAGATnnnnnnnnGTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG[Reverse primer sequence]

...which would mean Read 1, Index 1, and Read 2 sequencing primers are different, hence my confusion. Will either of these strategies work on the MiSeq? Has the above strategy worked for some folk? My thanks in advance.
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Old 07-04-2012, 01:20 AM   #20
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I think both should work.
The Illumina read primer is actually a primer mix of several primers. I guess there's probably primers for both sequences in there (TruSeq and Nextera).

However, if you're generating library directly from PCR, why not remove those sequences all together and just use custom read primers. Your custom read primers will just be versions of your fwd and rev primer sequences. You'll also waste less reagent by not sequencing your primers. See my post further up.
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