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Old 04-28-2008, 04:50 PM   #1
ScottC
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Default Illumina workflow, methods and equipment

Hi Everyone,

We're just getting our sequencer installed and running at the moment, so I'd be interested in talking with people who are either in the same position, or who already have theirs up and running. I'd be interested in starting some discussion on the methods that people are using for their sample prep, differences from the 'standard' illumina protocols, workflows and so forth. Is anyone else interested in sharing their experiences?

For example, what shearing method(s) are you using? Standard Illumina nebulisers, sonication, focussed acoustic shearing etc? I've heard a few people are ditching the nebuilsers because they can't get the fragmentation down to a small enough size, or that the yield is too low. What about cleanup of the DNA? Is anyone using SPRI, HPLC, or standard gels?

Cheers,

Scott.

Last edited by ScottC; 04-28-2008 at 05:22 PM.
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Old 04-29-2008, 01:27 PM   #2
ECO
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Great thread. I'd like to get a hold of the Illumina RNA tagging library prep protocols as a resource, as I've been asked multiple times.
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Old 04-30-2008, 07:59 AM   #3
apfejes
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I generally only deal with the post-lab stuff (eg. aligning, analysis, etc), though if you have specific questions, I can ask the lab people here who would know the answers.
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Old 04-30-2008, 09:09 AM   #4
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Here's a link to the Illumina DGE protocol
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Old 04-30-2008, 10:41 AM   #5
ECO
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Quote:
Originally Posted by pmcget View Post
Here's a link to the Illumina DGE protocol
Nice, thanks. Archived here as an attachment to the Illumina Tech Summary.

I also put up the companion protocol for the DpnII libraries as well**.


**special thanks to Google
Code:
{filetype:pdf site:arrayconsortium.tgen.org}
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Old 05-01-2008, 11:36 AM   #6
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We've been using an in-house developed sonicator and checking the fragments on an Agilent. For cleanup, you name it, we've used it. Some folks are using gels, I used magnetic beads for PCR amplicons and some others are using good old phenol/chloroform with alcohol extraction. All of these methods seem to give fine data, probably because of the size selection--which is on a gel anyway.

Cheers,
T
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Old 05-20-2008, 10:09 PM   #7
ScottC
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What about contamination control? Are sequencing groups taking any special measures to avoid it? How much of a problem is it?

Cheers,

Scott.
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Old 05-20-2008, 10:11 PM   #8
ScottC
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Quote:
Originally Posted by tesa View Post
Some folks are using gels, I used magnetic beads for PCR amplicons and some others are using good old phenol/chloroform with alcohol extraction. All of these methods seem to give fine data, probably because of the size selection--which is on a gel anyway.
Hi,

Thanks for the info.

Do you have any preferences amongst those? Any of the techniques give better results than the rest?

Cheers.
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Old 05-20-2008, 10:37 PM   #9
apfejes
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For contamination control, I don't think we do much for most types of sample at the BCGSC (though it's an ongoing project), but we definitely do with ChIP-Seq runs. We look for contaminating ladder sequences, and have (on occasion) tested our reads against various other possible sources such as bacteria or human genetic material.

As far as I know, only the ladder sequences has ever shown up in any volume - and we no longer use ladder sequences on the same gel as chip-seq samples because of it.

Anyhow, that's the little I do know.
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Old 05-21-2008, 08:34 AM   #10
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Default contamination control

I think generally, because after the sonication step to make the library you have to size select from a gel, the product should be very clean of other junk that might be in a DNA prep.

For bacterial contamination that might be in your original prep, we don't do anything specifically, but we do run our non-aligned reads against E. coli. to make sure we aren't getting a bunch of reads mapping to the E. coli genome.

Other than that, clean techniques is most of how we avoid contamination, currently.

Cheers,
Tesa
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Old 05-21-2008, 08:36 AM   #11
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I think many people just use a genomic DNA extraction kit and let the size selection on a gel step take care of the cleanup. Gel cleanup is probably the best way to get rid of small nucleic acids you don't want in your reaction. Plus, then there isn't an extra clean-up step, you can go straight to library making after you've extracted your DNA.

Cheers,
Tesa

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Hi,

Thanks for the info.

Do you have any preferences amongst those? Any of the techniques give better results than the rest?

Cheers.
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Old 05-21-2008, 02:10 PM   #12
ScottC
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Quote:
Originally Posted by tesa View Post
Other than that, clean techniques is most of how we avoid contamination, currently.

Yeah, that's what we'd planned. Nothing particularly special other than good technique, really. I've heard of several other labs, though, that have duplicated equipment, clean rooms, PCR hoods and other things like this. I don't know whether it's overkill. I guess it doesnt hurt if you have the equipment and facilities at your disposal.

Scott.
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Old 09-29-2008, 02:38 AM   #13
Jenny Russ
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I have a different question. Do you have experience with Solexa and longer fragments? I would like to know the maximum fragment length, where Solexa still works.

Jenny
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Old 09-30-2008, 03:48 PM   #14
apfejes
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works for what, Jenny?

Wouldn't that depend on whether it's a PET or a SET?
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Old 12-11-2018, 01:37 PM   #15
Alireza.Tafazoli
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Hey guys

Recently, our center provided the Illumina high throughput sequencing instrument (HiSeq4000) and I want to schedule a targeted paired-end sequencing on 300-350 interested genes (whole parts of those genes, including: promoter, 5' & 3' UTRs, exons and introns).
I want to know which Illumina library preparation kit would be the best choice for sequencing of such genes with different sizes.

Thanks for your help
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Old 12-13-2018, 05:13 AM   #16
tatisaccon
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Smile How important is to heat 5' Adapter in Step D of NEXTFLEX Small RNA-Seq Kit?

Hello!

How important is to heat 5' Adapter in Step D of NEXTFLEX® Small RNA-Seq Kit for Illumina® Platforms? In step D of the library preparation kit protocol I forgot to heat the 5 'adapter at 70C for 2 minutes and went straight to the incubation of 1h at 20 degrees C. The adapter 5 ' it's there, but it did not undergo this heating for 2 minutes. 12 samples in total. I wanted to know how much this can affect my final sample in sequencing, cause I do not have kit enough to do the 12 samples again.
If someone could share the experience with me would be great.

Thank you!!
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Old 12-13-2018, 11:33 PM   #17
nucacidhunter
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Heating denatures the oligo self-dimers and possible secondary structure. Skipping heating will reduce the available adapter for ligation. Its impact will depend on ratio of adapter to template RNA. If you have done recommended number of PCR cycles and obtained expected yield then it has not affected the results.
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Old 12-14-2018, 03:29 PM   #18
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Quote:
Originally Posted by nucacidhunter View Post
Heating denatures the oligo self-dimers and possible secondary structure. Skipping heating will reduce the available adapter for ligation. Its impact will depend on ratio of adapter to template RNA. If you have done recommended number of PCR cycles and obtained expected yield then it has not affected the results.
This helps. Thanks.
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Old 12-14-2018, 11:36 PM   #19
kayhand
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Hi,

Thanks for the info
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