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Old 08-30-2018, 02:12 AM   #1
M4TTN
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Default Cost-effective sequencing of >200 20kb amplicons

Dear all, title says it all:

We are looking into the best/most efficient/cost effective way of fragmenting/barcoding/sequencing a ~20kb target/fragment from ~200 independent sources. Final read depth needs to be ~1000x to enable variant calling.

Any ideas?

We were initially looking at NexteraXT, but this will come to over 7000 just for library prep materials for 200 samples.

Does anyone know of a homebrew option that enables multiplexing? Something with barcoded random primers perhaps?

Cheers! Matt
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Old 08-30-2018, 08:38 AM   #2
pmiguel
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I thought the Nextera XT kit had been discontinued in favor of the Nextera Flex kit?
Anyway, how about using Simone Picelli's Tn5 method for library prep?

Also, what about AmpliSeq? Now it is available on Illumina instruments...

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Old 08-30-2018, 09:32 AM   #3
M4TTN
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We were using NexteraXT for another project up until our stocks ran out. We've not tried Flex yet. Same cost though I think.

I'm not sure AmpliSeq would work for our question. We only have a single amplicon of ~20kb, but I suppose we could design primers to tesselate across it.

I can't imagine the technology is cheap though...coming direct from Illumina.

I did find another paper - we may try something related to this:

https://genome.cshlp.org/content/22/...expansion.html
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Old 08-31-2018, 03:03 AM   #4
pmiguel
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Don't forget the Picelli method:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4248319/
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Old 08-31-2018, 05:18 AM   #5
UCan'tBcereus
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Quote:
Originally Posted by pmiguel View Post
I thought the Nextera XT kit had been discontinued in favor of the Nextera Flex kit?
Anyway, how about using Simone Picelli's Tn5 method for library prep?

Also, what about AmpliSeq? Now it is available on Illumina instruments...

--
Phillip
Hi Phillip,

There was some confusion in our lab about this, but it was only the Nextera (not the Nextera XT) kit that was discontinued.

They also discontinued the Nextera XT Index Kit. Now they only sell the Set A/Set B/Set C/Set D Index Kits.

I have attached the original product obsolescence note from Illumina.

We are planning on trying to test Nextera Flex though, since automating the volumes in the kit down to 1/4 reaction will be more feasible than it is for Nextera XT. I don't know if you have figured your costs on full Nextera XT RXNS, but one of our customers routinely asks for 1/4 RXNS. We make successful libraries and they haven't complained about the data yet.

The one issue with Nextera Flex is that it only has 96 index combinations, while Nextera XT has up to 384.

Last edited by UCan'tBcereus; 08-31-2018 at 05:30 AM.
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Old 08-31-2018, 05:44 AM   #6
pmiguel
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Thanks UCan'tBcereus,
I had mis-read the PON.
You can just order oligos from IDT if you want all 384 combinations.
We already ordered a 48x48 set. Although since it is for the NovaSeq, it only suffices for 48 samples, not 2304.
I'm sure the XT index oligos would work for the Flex -- except that Flex may need a substantially higher amount. So it wouldn't be cost-efficient.
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Old 09-02-2018, 05:19 PM   #7
ikripp
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We have decided to move from XT to Flex and have found them comparable in cost and in output.
As Phillip suggested the XT index oligos work fine for flex, and I don't believe there is any difference in the amount required.
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