M2W FK Library prep, 6-14-17

KAPA Hyper Prep Kit – 250 ng per library (1 library)

1) End Repair and A-tailing

Qubit concentration of DNA pool: 23.7 ng/ul

Library:                          M2W-FK-6

ER and AT Buffer                 7 ul                                         *For 250 ng (250ng x 1ul/23.7ng = 10.5ul)

DNA (pool)*                         10.5 ul

ER and AT Enzyme              3 ul

H2O**                                  39.5 ul                                   **Tot vol must be 60ul (add H2O to get 60)

Total                                      60 ul

Incubate at 20C for 30 min, 65C for 30 min, 4C forever.

2) Adapter Ligation

NEXT FLEX ID:                      6 (CTTGTA)

H2O                                            5 ul

Ligation Buffer                         30 ul

Adapter (15 uM)*                     5 ul          *Adapter stock is 25 uM

DNA Ligase                               10 ul

ER and AT Product                   60 ul

Total                                        110 ul

* For 15uM adapter, solve for: (25uM)(x) = (15uM)(6ul) => 3.6 ul 25uM stock to 2.4 ul H2O = 6 ul 15uM stock

Incubate at 20C for 15 min.

3) Post Ligation Clean-up

M2W-FK-6

Adapter ligation product          110 ul

Ampure beads (0.8x)                 88 ul

Follow bead clean-up procedure. Elute in 35 ul.

Qubit: 5.74 ng/ul

4) Library Amplification

1x

2x HiFi                              25 ul

10x Primer                      5 ul

DNA Library                   20 ul

Total                               50 ul

 

Cycling parameters                                                             Cycles

Initial denature                            98C               45s                 1

Denature                                      98C               15s

Annealing                                     60C               30s                 2*

Extension                                     72C               30s

Final extension                           72C                1min              1

End                                                4C                 Forever

*Number of cycles chosen is based on amount of starting material (concentration of library pre-amp) and amount of final product needed. KAPA kit guidelines state that if starting with 100ng 3-4 cycles should produce 1ug. We started with ~115 ng (114.8 ng in 20ul – 5.74 ng/ul product). I chose 2 cycles (rather than 3-4) because we generally don’t need 1ug of amplified library, and 2 cycles has been sufficient in prior runs.

5) SPRI Clean-up

Library Amp Product             50 ul

Ampure beads (1x)                50 ul

Follow bead clean-up procedure. Elute in 35 ul.

Qubit: 10.7 ng/ul

M2W_FK_library

Above is the gel image of the library both before and after amplification.

The final library product was diluted to 10 nM (per sequencing facility instructions) and submitted for sequencing on 6-15-17.

M2W FK untagged and tagged pcr re-runs, 5-11-17

Re-ran technical replicates for 298. Did not get visible target amplification in last round of pcr, so hoping to get it in this round.

M2W_FK_51117_1_HS

No visible target band in above gel after untagged pcr.

M2W_FK_51117_2_HS

 

After tagged pcr, still no strong target band. Going to sequence these two technical replicates anyway to see if we can get any data from them. If not, the entire filter set for this time point will likely be removed from the analysis.

M2W FK PCR re-runs, 4-24-17

Re-ran 299 (both untagged and tagged) to get target product. In case I did not get target product, I also ran two untagged pcrs for 298 the filter replicate of 299 since I got target product for 298 previously.

M2W_FK_42417_HS

I still got only non-target product for 299 tag-3. I will next run tagged pcr for the 2 298 replicates.

M2W FK tagged pcr re-runs, 4-14-17

Re-ran tagged pcr for 14 samples that either had contaminated ntc’s or still showed strong non-target amplification in previous gels.

M2W_FK_41417_HS

 

Samples 398 tag-17, 325 tag-55, 326 tag-60, 327 tag-44, 298 tag-27, 299 tag-3 and 301 tag-11 will be re-run one more time to see if better product can be attained. I will also make and use new 1:10 dilutions in the hopes that this will improve the outcome. Additionally in cases where one of three replicates is not producing target product, I will make a technical replicate from one of the remaining filter extracts. I will also do this in cases where there are only 2 filter replicates. These will be sequenced in the instance that certain time points may have to be removed from the sequencing queue due to lack of target amplification.