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Old 08-01-2012, 06:58 AM   #1
AStretton
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Question MiSeq Newbie

Hello

We would like to use the MiSeq to sequence 8 amplicons in a few thousand samples. I realise that this is a low number of amplicons and this might cause us problems, we have been advised to use 3 primers sets for each amplicons to try to increase the diversity or to spike with 20% PhiX. Illumina doesn't support this sort study as such but has suggested their TruSeq HT kit. I was just wondering if anyone had any suggestions for alternatives? And if anyone could advise on doing this in a homebrew fashion we would be very grateful!

Thanks!
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Old 08-01-2012, 07:42 AM   #2
TonyBrooks
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How big are your amplicons?
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Old 08-01-2012, 11:50 PM   #3
AStretton
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They are 100-150bp
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Old 08-02-2012, 01:12 AM   #4
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Are you looking for alternative capture technology? if so Haloplex from Agilent is popular for small target regions (starting at 1kb) but I would expect PCR would be cheaper.
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Old 08-02-2012, 01:24 AM   #5
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I'll look into Haloplex. Thanks JPC!
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Old 08-02-2012, 02:31 AM   #6
AStretton
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Quote:
Originally Posted by JPC View Post
Are you looking for alternative capture technology? if so Haloplex from Agilent is popular for small target regions (starting at 1kb) but I would expect PCR would be cheaper.
Can you use FFPE extracted DNA with haloplex, do you know?
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Old 08-02-2012, 02:38 AM   #7
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I've read in Agilent publicity material that it's compatible;

http://www.agilent.com/about/newsroo...r-ca12021.html

I don't know of anyone who has tried yet

JPC
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Old 08-02-2012, 02:43 AM   #8
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Great! Thank you again JPC!
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Old 08-02-2012, 02:44 AM   #9
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Hi guys,
We were doing amplicon sequencing for the MiSeq. But we always spiked these samples into other runs to get the diversity needed.

Why not do the PCR with sequence-specific primer that contain a barcode on the 5' end. The diversity is needed especially in the first few (5 or 6, if I remember correctly) cycles. You could design like 16 different forward index-primers and 16 reverse index-primers which would allow post-analysis demultiplexing of 256 samples in a single run.

It is actually a bit tricky to get the adaptor sequences into the primers but if you rite me a PM I could send you the sequences we use (it's actually no secret, since they are based on this Illumina sequence letter...) for amplicon seq.

However, you need to demultiplex the samples lateron using a home-brew script, but that should be fairly easy...
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Old 08-02-2012, 03:00 AM   #10
JPC
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Hi AStretton, your profile says you are based in Cardiff, I assume you're on or near the Heath Park site? The NHS group there have a MiSeq, they may be able to help you out with other samples that may provide you with the diversity that your run will require?
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Old 08-02-2012, 03:06 AM   #11
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Hi JPC

Yes we do have a brand new MiSeq, unfortunately no one has actually used it yet and it therefore may not be possible to add in to other runs at the moment. We are just getting to grips with how everything works at the moment!
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Old 08-02-2012, 06:00 AM   #12
luckylove
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when do amplicon sequencing using Miseq some one adviced me to mix the samples with 50%Phix, then the results is good. i decide to do so. do you think it is fitable? thank you!


Quote:
Originally Posted by ulz_peter View Post
Hi guys,
We were doing amplicon sequencing for the MiSeq. But we always spiked these samples into other runs to get the diversity needed.

Why not do the PCR with sequence-specific primer that contain a barcode on the 5' end. The diversity is needed especially in the first few (5 or 6, if I remember correctly) cycles. You could design like 16 different forward index-primers and 16 reverse index-primers which would allow post-analysis demultiplexing of 256 samples in a single run.

It is actually a bit tricky to get the adaptor sequences into the primers but if you rite me a PM I could send you the sequences we use (it's actually no secret, since they are based on this Illumina sequence letter...) for amplicon seq.

However, you need to demultiplex the samples lateron using a home-brew script, but that should be fairly easy...
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