[kayhand]

Hi,

Thanks for the info

[nucacidhunter]

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.

[tatisaccon]

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!!

[Alireza.Tafazoli]

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

[apfejes]

works for what, Jenny?

Wouldn't that depend on whether it's a PET or a SET?

[Jenny Russ]

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

[ScottC]

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.

[tesa]

preferences

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

Hi,

Thanks for the info.

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

Cheers.

[tesa]

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

[apfejes]

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.

[ScottC]

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.

[ScottC]

What about contamination control? Are sequencing groups taking any special measures to avoid it? How much of a problem is it?

Cheers,

Scott.

[tesa]

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

[ECO]

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

{filetype:pdf site:arrayconsortium.tgen.org}

[pmcget]

Here's a link to the Illumina DGE protocol