Rapid Libraries

[nickloman]

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.

[LMcSeq]

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!

[nickloman]

Great stuff. We're really looking forward to moving to them as the current workflow is still a big pain for us.

[AKroxy]

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

[LMcSeq]

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!

[pmiguel]

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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Phillip

[Old guy]

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?

[pmiguel]

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

--
Phillip

[Old guy]

Don't quite understand

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?

[LMcSeq]

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.

[pmiguel]

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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Phillip

[RCJK]

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.

[JohnG]

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

[RCJK]

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

[Old guy]

Flourimeter question

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.

--
Phillip

Do you use different standards to quantitate based on the GC content of the genome or based on the size of the genome?