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Old 02-01-2012, 06:38 AM   #1
Rocketknight
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Default Cutting corners with Illumina Sample Prep

Hey all, just a quick question regarding Illumina's TruSeq DNA sample prep protocol. After adapter ligation, there's a cleanup step with Ampure XP. If you're doing the gel-cut size-selection step though, that step comes directly afterwards. The manual seems to suggest that you need to do the Ampure cleanup even if you're planning to do the gel-cut afterwards, but I feel like electrophoresis should remove any contaminants well enough on its own.

Has anyone tried skipping straight to electrophoresis without the Ampure cleanup? Is there any reason that shouldn't work?
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Old 02-01-2012, 06:55 AM   #2
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I just skip the electrophoresis altogether, having sized the population with AMPure before the ligation.

The AMPure prior to the gel is likely to ensure that a majority of the ligase and free adapter (not to mention buffer/salt/DTT/ATP) are removed before sizing the sample. That much protein will almost certainly alter migration patterns...but by how much I can't say. It's an easy experiment to do though.
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Old 02-01-2012, 07:23 AM   #3
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Agree with ECO you need to do the AMPure purification before you run it on a gel. You are isolating small DNA fragments and you can't run the gel for very long or your gel slice will be too big. The salt front will likely run up against were you want to cut. If you want to cut corners, skip the gel purification.
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Old 02-01-2012, 08:34 AM   #4
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Ah, good points, both of you. I'm afraid I'll have to keep the gel-cut size-selection step though. I'm using a Bioruptor rather than a Covaris and I'm doing exome enrichment followed by paired-end sequencing. I'm worried that if I allow big inserts through I'll end up getting a lot of reads that lie outside exons. Is that just paranoia?
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Old 02-01-2012, 09:52 AM   #5
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Quote:
Originally Posted by Rocketknight View Post
Ah, good points, both of you. I'm afraid I'll have to keep the gel-cut size-selection step though. I'm using a Bioruptor rather than a Covaris and I'm doing exome enrichment followed by paired-end sequencing. I'm worried that if I allow big inserts through I'll end up getting a lot of reads that lie outside exons. Is that just paranoia?
If you sonicate on high for long enough I think you can get the Bioruptor size distribution down to a suitable size. May be worth a try.
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Old 02-01-2012, 10:46 AM   #6
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I can definitely get a peak in the size range I want, my main problem is that I feel the distribution will be broader than with Covaris, and so I'll get a lot of undesirably large inserts, even if the peak is in the right place.
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Old 02-01-2012, 11:19 AM   #7
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Look into double sided AMPure cuts to avoid the gel....we do it all the time. They are described here and in the literature... I can't find the links now but that should get you started.
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Old 02-01-2012, 02:15 PM   #8
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Aha, awesome. I didn't realize bigger fragments outcompeted smaller ones when the beads were limited, I thought they just magically didn't bind fragments below a certain size or something. Is there a standard table for what bead:dna ratio I should use for each size cutoff, or should I determine my own?
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Old 02-02-2012, 07:43 AM   #9
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Quote:
Originally Posted by Rocketknight View Post
Aha, awesome. I didn't realize bigger fragments outcompeted smaller ones when the beads were limited, I thought they just magically didn't bind fragments below a certain size or something. Is there a standard table for what bead:dna ratio I should use for each size cutoff, or should I determine my own?
The beads themselves are not the issue, are they? I understood that AmpureXP is a PEG cut with a fancy way to recover the precipitant, right?

Except I guess the beads themselves do have a maximum binding capacity. Anyone know what that is?

Oh, never mind, that is all covered here.
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Old 02-02-2012, 09:20 AM   #10
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Ahh, my bad again. It's the concentration of PEG in the Ampure XP buffer that governs what fragment size will precipitate. Sorry for the confusion.
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Old 02-02-2012, 09:40 AM   #11
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This paper has some information on salt and PEG concentrations to size select using AMPure beads. They used a Bioanalyzer too.
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Old 05-10-2012, 07:57 AM   #12
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Thanks all for the replies! I just started another batch of samples and got really nice size selection with Ampure. I sheared with a Bioruptor down to a peak size of about 260bp, then did the standard Illumina prep up until the first cleanup after end repair, then replaced their Ampure cleanup with the following:

1) Add 90ul Ampure XP to the 100ul end-repaired DNA.
2) Incubate 20 minutes
3) Separate beads and take supernatant
4) Add 20ul Ampure XP to the supernatant
5) Incubate 7 minutes
6) Separate the beads and clean them as normal (ethanol wash, dry, resuspend, etc)

This got me pretty much the perfect size range for 2x100bp exome sequencing. The only problem is I estimate I lost about 80-85% of my input DNA in the process. I have plenty of sample DNA to work with, but I'd prefer to only do the size-selection on DNA I've end-repaired. Does anyone know how well the Illumina end repair mix works if you use 4ug of sheared DNA at that step instead of 1ug?

Also, Bioanalyzer trace for the Ampure size-selected DNA:

Last edited by Rocketknight; 05-10-2012 at 08:13 AM.
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Old 05-16-2012, 01:35 PM   #13
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Hi Rocketnight,

Did you by any chance do a Qubit analysis after you shearing to see if you are losing most of your DNA as single stranded DNA as a result of high energy over-shearing?
Comparing the Qubit results of your unsheared sample to the sheared sample will let you know if that is the cause of high loss of available sample for library preparation.

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Old 05-16-2012, 01:41 PM   #14
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I checked that, yeah. I didn't lose much DNA in shearing - I think it just gets lost during double-Ampure selection. Do other people lose much less DNA with it?
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Old 06-26-2012, 09:01 AM   #15
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You might recover a bit more DNA if you reduce the first bead incubation time to 10 minutes. It is my impression that the size selection is anything but absolute - i.e. with longer incubation more DNA will keep on precipitating.
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Old 06-28-2012, 03:44 AM   #16
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Hey, sorry to dredge this thread up again, but I have another question! Illumina recommend a double-Ampure cleanup after ligation with a 1:1 ratio of Ampure. This confused me way back when I started this thread, but now I realize that as well as cleaning up the reaction this is also a way of removing DNA below about 140bp - adapters and adapter dimers, in other words.

So here's my next question: Can I save time by doing a different cleanup at this step (e.g. QIAQuick PCR Purification) if I'm going to do downstream exome capture? I'm assuming that the exome capture step will be largely unaffected by the adapter dimers. Or have I overlooked something?
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Old 06-28-2012, 04:30 AM   #17
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Small amounts of adapter dimers will probably not be too damaging, but how much time do you really save by doing a QIAQuick PCR Purification over an Ampure XP Bead cleanup? It can't be more than 5 minutes. Is that worth deviating from the protocol when you are going to do a capture for probably at least 24 hours?
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Old 06-28-2012, 04:46 AM   #18
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It's just me being lazy, but two consecutive Ampure cleanups take about an hour and a half with Illumina's recommended incubation and drying times, compared to about ten minutes to column-purify.

But yeah, you're right. If there's any uncertainty I'll stick with the Ampure method.
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Old 06-28-2012, 04:52 AM   #19
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I don't know what Illumina recommends, but it's probably unnecessary. Mix beads with sample, vortex vigorously, 5 minute incubation, pellet beads with magnet, discard supernatant, add in 70-80% EtOH (enough to cover the beads), discard the EtOH, repeat the wash, discard the EtOH, suck out residual EtOH at the bottom of the tube with a fine-tipped pipette, place tube in heat block for a few minutes, add in water/TE to elute, vortex and incubate for 2 minutes, then transfer out. Whole process never takes me more than 20-25 minutes if I'm trying to get done quickly and only am working with a few tubes.

Another idea (that I have not tried) is to mix in the beads for the first cleanup, do the vortext/5 minute incubation step, discard supernatant, do one wash with 70-80% EtOH, discard the EtOH, and then directly add in the beads for the second cleanup and continue from there.
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Old 06-28-2012, 05:43 AM   #20
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Awesome, but do you lose much DNA from the shorter Ampure incubation time? I feel like not all of it would have precipitated after 5 minutes.
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