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Old 01-26-2010, 06:04 AM   #1
nickloman
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Default Rapid Libraries

Hello

Has anyone got and started using rapid libraries for 454?

Is anyone in a position to test the claim that 500ng of starting material is now sufficient?

Cheers

Nick.
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Old 01-26-2010, 09:43 AM   #2
LMcSeq
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I've prepped 5 rapid libraries so far and its so fast and easy. Based on their metrics of success, the 500ng input has been sufficient. However, we haven't yet sequenced the libraries. I will taking them forward through the rest of the process over this and next week. Will let you know how it works out!
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Old 01-29-2010, 02:13 AM   #3
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Great stuff. We're really looking forward to moving to them as the current workflow is still a big pain for us.
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Old 02-09-2010, 11:39 AM   #4
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Hi LMcSeq
Have you gotten your rapid library results yet? How were they?
i'm prepping a cDNA rapid library for sequencing this month. This is my first nextgen attempt, so I'm nervous!
thanks
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Old 02-09-2010, 11:45 AM   #5
LMcSeq
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I have my results! Everything came out great. Got over 2 million raw wells and 339 million+ bases.

If you need help, drop a line! Good luck!
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Old 02-10-2010, 11:59 AM   #6
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The rapid cDNA libraries work well for us. Two libraries we made from 200 ng (each) polyA RNA were run as a region each. We also got 339 million bases total. Not our best run, but not bad. We also got about 175 megabases of sequence from 5/8ths of a PTP comprising 3 MID rapid cDNA libraries.

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Old 02-10-2010, 12:04 PM   #7
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Are the results of the Rapid library starting with 500ng consistent? Another question is what kind of results has anybody had with the Titanium library kits? Lastly is there another location on this forum that address library construction trouble shooting?
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Old 02-10-2010, 01:16 PM   #8
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Quote:
Originally Posted by Old guy View Post
Are the results of the Rapid library starting with 500ng consistent? Another question is what kind of results has anybody had with the Titanium library kits? Lastly is there another location on this forum that address library construction trouble shooting?
We only made one DNA (shotgun) rapid library. It failed miserably because the DNA concentration was 1000x low. But I don't think there should be any problems. The 10 ug asked for in the normal protocol was ridiculously high--probably because of the library binding/ssDNA elution step.

The Titanium library kits seemed to work okay. But sometimes we would get lots of adapter dimers and need to gel isolate the libraries to avoid those.

If you want to troubleshoot 454 library construction, this is the forum to use. If you have more general questions you could post in one of the application forums:

http://seqanswers.com/forums/forumdisplay.php?f=9

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Old 02-11-2010, 06:50 AM   #9
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Default Don't quite understand

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Originally Posted by pmiguel View Post
We only made one DNA (shotgun) rapid library. It failed miserably because the DNA concentration was 1000x low. But I don't think there should be any problems. The 10 ug asked for in the normal protocol was ridiculously high--probably because of the library binding/ssDNA elution step.

The Titanium library kits seemed to work okay. But sometimes we would get lots of adapter dimers and need to gel isolate the libraries to avoid those.

If you want to troubleshoot 454 library construction, this is the forum to use. If you have more general questions you could post in one of the application forums:

http://seqanswers.com/forums/forumdisplay.php?f=9

--
Phillip

You said "We only made one DNA (shotgun) rapid library. It failed miserably because the DNA concentration was 1000x low. But I don't think there should be any problems." So do you feel that the rapid libraries will work with 500 ng starting material?. Is this starting material quantity based on quantification before or after nebulization?
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Old 02-11-2010, 07:40 AM   #10
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We made 5 libraries with 500ng starting material each. It worked great for all, but we use Covaris for shearing so we don't lose material in the nebulization step. We modified the MinElute protocol immediately following the shearing to scale down to the 120ul that comes off the Covaris vs. the 600ul that comes out of nebulization. The results were very consistent between the libraries--I even had two libraries that came out with the exact same molecules/ul. I do think that I need to modify my shearing protocol a bit to add more time because the average fragment lengths were on the higher side (upper 800s). Although the kit says that 600-900bp average is good, I'd rather it be a bit smaller so that there is limited kick out of larger fragments in emPCR to prevent preferential amplification of only smaller frags.
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Old 02-11-2010, 01:36 PM   #11
pmiguel
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Quote:
Originally Posted by Old guy View Post
You said "We only made one DNA (shotgun) rapid library. It failed miserably because the DNA concentration was 1000x low. But I don't think there should be any problems." So do you feel that the rapid libraries will work with 500 ng starting material?. Is this starting material quantity based on quantification before or after nebulization?
Before. But use a fluorimeter to quantitate. DNA preps, especially genomic DNA preps are often >90% RNA (sometimes degraded to short pieces with RNase). Or phenol! Phenol absorbs strongly at 270 nm. So if you use UV spectrophotometry, make sure your peak is at 260, not 270.

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Old 02-11-2010, 05:23 PM   #12
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I've done 15 rapid libraries, some w/ MIDs and some with out and they worked well with 500ng starting material. The rapid protocol is much faster than the general library prep.
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Old 02-14-2010, 02:49 PM   #13
JohnG
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Hello,

RCJK which type of MID tags have you used for rapid library preparation? Have you tried to use MID tags from standard library protocol?

I've asked Roche support about using MIDs from standard protocol for preparing Rapid library, but they told me that it is not recommended.
I think that they probably changed primers concentration used in adaptor ligation step. Both standard and rapid MIDs can be used later in emPCR so primers sequence is probably the same.

Cheers

John
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Old 02-14-2010, 06:54 PM   #14
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Hi JohnG,
I used the Rapid Library MID adaptors provided by Roche. They provide a kit with 12 MIDs. The RL adaptors and MID adaptors are different than those of the general library prep. They are now Y adaptors labeled with FAM (I believe) for easier quantification at the end of the protocol.

Cheers
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Old 02-16-2010, 06:58 AM   #15
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Default Flourimeter question

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Originally Posted by pmiguel View Post
Before. But use a fluorimeter to quantitate. DNA preps, especially genomic DNA preps are often >90% RNA (sometimes degraded to short pieces with RNase). Or phenol! Phenol absorbs strongly at 270 nm. So if you use UV spectrophotometry, make sure your peak is at 260, not 270.

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Do you use different standards to quantitate based on the GC content of the genome or based on the size of the genome?
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Old 02-16-2010, 07:51 AM   #16
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Do you use different standards to quantitate based on the GC content of the genome or based on the size of the genome?
No. Most of the time these figures are "ballpark" anyway. 50% high or low will make little difference.

Phenol or RNA, though, can easily contribute >90% of the UV absorbance of a genomic DNA sample. 10-fold low on the amount of input DNA does make a difference.

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Old 03-04-2010, 05:50 AM   #17
LMcSeq
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Hi all,
Quick question: I'm finding the my average frag length on the high sensitivity chip at the end of the rapid protocol is on the larger side. My sequencer results have had low average fragment lengths. I'm wondering if the larger frags aren't being amplified because they're too big for the microreactors, causing preferential amplification of smaller frags. I'm using the Covaris settings recommended for 500bp average. Anyone else having any similar issue?
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Old 03-04-2010, 08:03 AM   #18
pmiguel
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Quote:
Originally Posted by LMcSeq View Post
Hi all,
Quick question: I'm finding the my average frag length on the high sensitivity chip at the end of the rapid protocol is on the larger side. My sequencer results have had low average fragment lengths. I'm wondering if the larger frags aren't being amplified because they're too big for the microreactors, causing preferential amplification of smaller frags. I'm using the Covaris settings recommended for 500bp average. Anyone else having any similar issue?
It could be. Typical PCR reactions yield vastly more product for short templates than for longer ones.

But it could also be that your read lengths are short for some other reason. If your templates are actually short you should have a high number in your "Short Primer" metric. If not, then something else is likely the source of your short reads.

I will mention one: polyA, of course, is the bane of 454 runs...

Even with random primed cDNA libraries, it is possible to run into polyA problems. It should be less, but polyA+ RNA isolation will enrich for polyA. If you start with heavily degraded RNA and pull out polyA from that, you may get a high percentage of your cDNA being polyA or polyT--even if you used random primed reverse transcription.

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Old 03-04-2010, 08:09 AM   #19
LMcSeq
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Hi Phillip,
Thanks for your reply. We've had our 454 FAS out and he said that its likely a problem in library prep or emPCR based on the output from the support tool (which customers can't view themselves). He said the short primer is in spec.

We are shearing whole genomic microbial DNA, not cDNAs. I think that was another contributor to this thread.
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Old 03-04-2010, 08:47 AM   #20
pmiguel
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Quote:
Originally Posted by LMcSeq View Post
Hi Phillip,
Thanks for your reply. We've had our 454 FAS out and he said that its likely a problem in library prep or emPCR based on the output from the support tool (which customers can't view themselves). He said the short primer is in spec.

We are shearing whole genomic microbial DNA, not cDNAs. I think that was another contributor to this thread.
Okay, but if short primer is in spec, that means you do not have a large number of reads that traverse the insert completely. So preferential amplification of your small templates is not the issue.

It could be that you have too many templates longer than the maximum optimum amplification length of the emPCR kit (~1 kb, I think). Those would tend to produce lightly templated beads that might give you short reads.

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