Optimal library concentration for sequencing
Материал из Zbio
NB! pre-PCR operation
[править] Real-Time PCR
NB! measurement of library concentration by Real-Time PCR should be performed as close as possible to loading. Repeat measurement, if the last one was done more than 3 monthes ago 
- calculate the number of RT PCR reactions: "N of samples" + positive control + negative control;
- prepare RT-PCR common mix in ice-bath:
* SYBR Green PCR core reagents kit from ABI, #4304886
** use following primer pairs:
- » library for SR: "PCR primer 2.1" and "adapter primer 1"
- » library for PE: "#611" and "adapter primer 1"
*** Bioline #BIO-21046. It is also possible to use AmpliTaq Gold, 5u/µl; ABI, but PCR efficiency and yield will be lower
- distribute RT mix to 100µl optical RT-PCR tubes/strips: 19µl in each tube;
- store RT-PCR tubes on ice;
- put back RT PCR reagents;
- prepare libraries (up to 20nM):
--- in post-PCR room ---
- » melt libraries
- » wortex
- » spin down
- » if necessary, measure concentration and prepare 10nM dilution
- » install library tubes into tube-holder
- » bring tube-holder into post-PCR room, put it on a piece of paper towel
- add 1 µl of library solutions to RT tubes;
- wash, or put on new gloves;
- close tubes/strips tightly, mix by finger tapping, spin down;
- start PCR program :
- 95°C, 10:00
- 95°C, 0:15
- 65°C, 0:30
- 72°C, 0:30
- go to 2, 39 times
- melt curve analysis
- bring library tubes back to post-PCR room:
- » snap freeze them in liquid nitrogen
- » store at -20°C
- determine the optimal loading concentration for each library
[править] Library stock preparation
- (if not already done) determine library concentration on Qubit: HS kit;
- if library concentration is more, than >20nM prepare 10nM dilution in LSB;
[править] Optimal concentration for loading
should be done after "first base report"
- on the basis "loading concentration" and "number of clusters per tile" from the first base report calculate the optimal loading concentration:
- insert it as a coment into library database
- LSB: EB, 0.1% Tween20
- » high sensitivity
- » detection of only amplifiable sequences
[править] Open questions
this protocol uses different "reference libraries" for optimization of library loading. It may lead to "double mistakes"
- » one occasional mistake during library loading (for example, pipetting mistake)
- » would lead to the "calculation mistake" for the next library.
It is worth to prepare a large amount of "reference standard, aliquote it, and use as a "stable reference". It also gives a possibility to control RT PCR itself, because "stable reference" should rize on the same cycle.
[править] See also
- Buehler B, Hogrefe HH, Scott G, Ravi H, Pabo'n-Pen~a C, O'Brien S, Formosa R, Happe S. Rapid quantification of DNA libraries for next-generation sequencing. Methods. 2010 Apr;50(4):S15-8.
- qPCR for quantification of NGS libraries
- ↑ for low concentrated libraries concentration may decrease upon storage or repeated thawing/freezing cycles
- ↑ RT PCR controls:
- positive control: <20nM stock (freshly prepared every month) of previously sequenced library, for which the optimal loading concentration is known
- negative: H2O, mQ
- ↑ PCR primer 2.1: #601, 5’ - CAAGCAGAAGACGGCATACGAGCTCTTCCGATCT - 3’
- ↑ 4,0 4,1 adapter primer 1: #603, 5’ - TACACTCTTTCCCTACACGACGCTCTTCCGATCT - 3’
- ↑ #611: partly complementary to PE adapter primer 2, 5' – GGCATTCCTGCTGAACCGCTCTTCCGATCT - 3'
- ↑ library stock may cause contamination. Tube holder from post-PCR room should not contact things (table) in the pre-PCR room
- ↑ artificial hot start: start PCR program, pause on 95°C, install tubes and resume pause
- ↑ do not use final 5-10 min 70-72°C extension. In this protocol it is completely useless
- ↑ optimal loading concentration for the reference library should be calculated on the basis of real sequencing