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Old 09-08-2016, 03:31 AM   #1
Lovro
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Default 1000bp insert size with Illumina TruSeq DNA PCR-Free Library prep kit

Hi!

I'm considering modifying Illumina TruSeq DNA PCR-Free Library prep kit protocol to produce 1000bp insert size libraries for sequencing on MySeq.

The reason is that the libraries produced will be enriched with custom DNA probes. With larger insert size I plan to get more DNA pulled and sequenced per probe (custom probes are expensive).

We have sequenced Nextera made libraries of such insert size on MySeq without any problems with sequencing before.

I was wondering, if anybody has experience with modifying Illumina TruSeq DNA PCR-Free protocol and would be willing to share his/her experience.

It seems like the logical steps would be to change Covaris settings to produce 1000bp fragments, change the starting concentrations (perhaps from 2ug for 550bp to 3ug), and change the concentrations of SPB in cleanup steps.

Also, since DNA will later be enriched, perhaps fewer cleanup steps would be needed (to preserve sample DNA) as shorter fragments without insert would not be pulled anyhow? Please correct me if I'm wrong.

Thank you,
Lovro
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Old 09-08-2016, 03:30 PM   #2
luc
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Your protocol modifications sound reasonable. I do not have experience with this specific kit.
Did your previous reads from such libraries indeed represent 1kb inserts according to alignments?
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Old 09-08-2016, 05:54 PM   #3
Brian Bushnell
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I'd also be very interested in seeing an insert size distribution of your prior 1kb libraries, and how you size-selected, if you did anything special. I was under the impression that such large inserts caused problems in bridge amplification and were out-competed by shorter fragments, but I have not really seen any hard data. By "Nextera" I assume you mean normal Nextera libraries, not long-mate-pair, right?
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Old 09-09-2016, 09:28 AM   #4
Lovro
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I'm sorry, but I was misinformed.

Indeed the libraries are 1000bp long on average, but after sequencing these are the results:



Yes, I meant normal Nextera lib and loading concentrations and enzyme quantity are the only parameters that had been changed.
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Old 09-09-2016, 09:51 AM   #5
Brian Bushnell
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That looks more like I would expect Although you can shift the curve toward longer inserts with size-selection. Sorry, I can't offer any specific advice on library prep.
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Old 09-09-2016, 12:18 PM   #6
milw
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Quote:
Originally Posted by Brian Bushnell View Post
I'd also be very interested in seeing an insert size distribution of your prior 1kb libraries, and how you size-selected, if you did anything special. I was under the impression that such large inserts caused problems in bridge amplification and were out-competed by shorter fragments, but I have not really seen any hard data. By "Nextera" I assume you mean normal Nextera libraries, not long-mate-pair, right?
I think that's what the graph is showing- clusters can form only from the smaller fragments in the distribution so very few show inserts of >600 bp.You'd really need to make a mate pair library to see the ends of 1000bp inserts.
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Old 09-30-2016, 03:19 PM   #7
Dongmei_MPI
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Default Fragement size --TruSeq Nano librarry prep.

Sorry to bother everyone.

I have problem in preparing library for Miseq, and need everyone's help. I have used the TurSeq LT library prep Kit for a few samples, the DNA was restriction digest with Ecor I (ezRAD method) and then followed the instructions form the manual. However the fragments are much bigger than expected. The average size is around 1000 bp, not 500-700bp expected. I have used the beads in the kit to remove the bigger and small sizes

Any suggestions on how to improve the library? Thank you,

Last edited by Dongmei_MPI; 10-01-2016 at 10:32 PM.
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Old 09-30-2016, 03:59 PM   #8
luc
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Better open a new thread for your question. Are you perhaps contradicting yourself regarding the sizes? Could you upload your bioanalyzer traces?
Your 100 bp sized fragments could be adapter dimers (120 bp) - indicating that you did not have enough digested DNA sample?
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Old 09-30-2016, 03:59 PM   #9
nucacidhunter
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I am not sure if you mean the library size was smaller than you expected. If you follow TruSeq protocol you have options of selecting fragments with average 350 or 550 bp. TruSeq instructions are for randomly sheared DNA fragments to a particular peak size but restriction fragments can have different size distribution depending on species and choice of restriction enzymes. This will affect the final library insert peak size so you will have to find suitable restriction enzyme by trial and error or by in silico digest.
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Old 10-01-2016, 10:29 PM   #10
Dongmei_MPI
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Default ~1000bp, not 100 bp

Sorry, the average sizes are around 1000 bp, not 100 bp.

That was my first post, I tried to post a new thread, but I could not find a tab to do so.

I saw this topic is talking about ~000 bp insert size, so I posted my one.

Last edited by Dongmei_MPI; 10-01-2016 at 10:31 PM.
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Old 10-02-2016, 12:41 AM   #11
nucacidhunter
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Link for how to start a new thread:

http://seqanswers.com/forums/showpos...69&postcount=9

You need to optimise size selection. To reduce large fragments increase bead ratio that you add at the start. That would take out more of larger fragments.
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Old 10-02-2016, 01:11 AM   #12
avo
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Adapting TruSeq PCR free for approx. 1000 bp insert size is possible. The following plot is from a library size selected on a BluePippin after adapter ligation.

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Old 10-03-2016, 08:32 AM   #13
pmiguel
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Quote:
Originally Posted by avo View Post
Adapting TruSeq PCR free for approx. 1000 bp insert size is possible. The following plot is from a library size selected on a BluePippin after adapter ligation.

Note that if you cluster this library I would expect the majority of your insert sizes to be below 750bp. The shorter amplicons compete much more effectively for clustering than the longer ones.

See my previous thread for some detail:

http://seqanswers.com/forums/showthread.php?t=20839

--
Phillip
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Old 10-03-2016, 11:10 AM   #14
BHR
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For OP:

1. what is your target size? It may require quite a lot start material w/o pre-capture pcr and post-capture pcr, supposedly that's what you want. We have tried several protocols/workflow for making 2kb insert capture library, with or without pcr. Very limited amount library was produced with pcr-free protocol for a 15mb target panel.

2. You can try covaris shearing for 1kb and bluepippin or you can tagmentation or fragmentase, these non-covaris shearing gave more library yield

basically it's doable, but you will need a lot starting material

Last edited by BHR; 10-03-2016 at 11:34 AM.
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