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Old 04-01-2013, 12:03 PM   #1
JChase
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Default Generating larger insert sizes (>300bp) using TruSeq RNA protocol

Hello,

I'm hoping to get some advice on using the TruSeq RNA kit to produce libraries with 300-350bp insert sizes. Has anyone here found a reliable way to do this with minimal modifications to the TruSeq protocol?

In Appendix A of the protocol, Illumina provides some alternate fragmentation protocols. The most straightforward seems to be decreasing the library insert fragmentation time (the time at 94 degrees). On another post on this forum, a user provided bioanalyzer traces of final library products using 0, 4, and 8 minute fragmentation times (link: http://seqanswers.com/forums/showthread.php?t=13744). They were somewhat surprised to find that at a 0 minute fragmentation time, their final library product demonstrated fragment sizes of over 1kb (compared the Illumina's predicted 130-350bp fragment size). I spoke with Illumina support today in hopes of getting some more insight about this. The person I spoke with said that because of random hexamer priming, the fragments should be relatively small regardless of the size of the fragmented mRNA; she also suggested that perhaps the long RNA product was somehow contaminating the bioanalyzer trace. I don't know enough about bioanalyzer DNA chips to know if that's a possibility. She also said it was very important to let the sample incubate at 4 degrees for a couple of minutes even when using a 0 minute fragmentation time at 94 degrees, and I was unsure if this was the case in the post I linked to earlier.

One of the things I have considered trying is using a 20 second insert fragmentation time. Illumina unfortunately has no data to share about using fractions of a minute in this stage of the protocol. Another possibility I suppose would be to elute the mRNA before fragmentation, reverse transcribe with poly-A primers, and then fragment using our Bioruptor. I'd prefer to avoid that if possible, simply because we have never used it to shear cDNA and we would also have to optimize the bioruptor for the concentrations and quantities produced by the TruSeq protocol.

Any suggestions would be appreciated! Thanks!
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Old 04-03-2013, 06:03 AM   #2
pmiguel
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Hi JChase,
Okay, this is relatively simple, but Illumina just doesn't seem to get it. Think of it in two stages:

(1) Your maximum cDNA length will be determined by the amount of time you heat degrade your RNA.
(2) Your minimum cDNA length will be determined by the ratio of sample:Ampure you use in one or all of your clean-ups.

Upon clustering minimum size trumps maximum size. Look at your final bioanalyzer trace of your library. If you have short amplicons trailing off to the left, then you will mainly see those upon sequencing. You can think of them as queuing up for spaces on the flow cell -- shortest amplicon gets in line before longer ones.

So, again, two (2) parts to this. If you want to sequence longer cDNA fragments you must
(1) reduce your fragmentation time

and

(2) you must size select for the larger RNA, cDNA and/or amplicons.

We use lower ampure amounts to do this.

It really is that simple. The Illumina TruSeq RNA kits kick ass -- they get higher polyA yields than any other kit we have tried and are capable of producing >2kb cDNA fragments, if needed. To take advantage of this, you just do the most obvious things -- lower the amount time you fragment ***and*** get rid of the stuff smaller than what you want to sequence so the longer stuff can be sequenced.
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Old 04-03-2013, 10:45 AM   #3
JChase
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Thanks for the response. I was anticipating doing some size selection using our Pippin Prep, but I'm still left wondering about the best fragmentation time to get as much of the cDNA as possible around the 300bp size. I understand that the smaller fragments end up being sequenced and that you need to get rid of things below a certain size, but isn't it also important to try to get the majority of the cDNA into the range that will be sequenced? It just strikes me as a bit wasteful, the idea that you would make a library (like the one in the link I posted above) where the vast majority of inserts are so large that they'll not be sequenced. I suppose I should just give 4 minutes fragmentation time a shot and see how it goes.

Thanks again.

Edit: I also found that the NEWEST version of the protocol provides some additional information about alternative fragmentation conditions.

Last edited by JChase; 04-03-2013 at 05:17 PM.
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Old 05-15-2013, 08:59 AM   #4
cfielding1
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Red face RNA insert size

Hi pmiguel,

I was reading your post and am currently working on a project to increase the insert size of RNA for the TruSeq RNA kit to 500bp or more. It sounds like you have already managed to get sizes as large as >2kb from reducing the fragmentation time and by using lower amounts of ampure beads.

I was wondering

(1) We have a covaris and have sheared our library using 30s, have you used less fragmentation time than this?
(2) There are many different stages that use an ampure bead clean up within the processing a)second strand cDNA (90ul), b)End Repair (160uL), c) Ligate Adapters (42uL), Ligate Adapters (50uL), d) Enrich DNA fragments (50uL). Have you reduced the volume for all stages or just one in particular.
(3) How much have you reduced the volume of Ampure beads? How much should we reduce it by to get a library of around 500bp, 1000bp or 2kb+?

Any help you could give me would be much appreciated.

Claire
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Old 05-16-2013, 05:36 AM   #5
pmiguel
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Quote:
Originally Posted by cfielding1 View Post
Hi pmiguel,

I was reading your post and am currently working on a project to increase the insert size of RNA for the TruSeq RNA kit to 500bp or more. It sounds like you have already managed to get sizes as large as >2kb from reducing the fragmentation time and by using lower amounts of ampure beads.

I was wondering

(1) We have a covaris and have sheared our library using 30s, have you used less fragmentation time than this?
Good luck,

--
Phillip
So this is a non-TruSeq RNA library prep? You don't need to sonicate with most RNA preps because you can heat/divalent cation fragment the RNA.
But, just look at your Covaris manual, it has programs to generate various sizes.
Quote:
Originally Posted by cfielding1 View Post
(2) There are many different stages that use an ampure bead clean up within the processing a)second strand cDNA (90ul), b)End Repair (160uL), c) Ligate Adapters (42uL), Ligate Adapters (50uL), d) Enrich DNA fragments (50uL). Have you reduced the volume for all stages or just one in particular.
We usually reduce it for all steps. But we generally will keep the PEG-containing supernatants around for a time in case something goes terribly wrong.
Quote:
Originally Posted by cfielding1 View Post

(3) How much have you reduced the volume of Ampure beads? How much should we reduce it by to get a library of around 500bp, 1000bp or 2kb+?
You would want to calibrate your Ampure batch probably. Also, I think you would need to use some other size fractionation for getting above 1000bp. But who knows?
We just calibrated our most recent batch of ampure. Here is what we got:

Quote:
Originally Posted by cfielding1 View Post
Any help you could give me would be much appreciated.

Claire
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