AlanFung:LabNotes/2015/2015-7-14: Difference between revisions
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==Post-PCR SPRI== | ==Post-PCR SPRI== | ||
* (1:0.8) 40ul beads elute with 20ul Low EDTA TE. | * (1:0.8) 40ul beads elute with 20ul Low EDTA TE. | ||
[[File:ZhangLab_2 2015-07-15 23hr 17min.jpg]] | [[File:ZhangLab_2 2015-07-15 23hr 17min.jpg|400px]] |
Revision as of 09:54, 16 July 2015
Swift Methyl-Seq DNA Library Prep & Capturing
- We are interested in the performance of the Swift Bioscience Methyl-Seq DNA library kit
- Since we have WGB libraries made using Kapa we can compare swift against it.
- 2nd Experiment was done by Noi (Kapa Hyper Lib. Prep + Seqcap) on Noi/NOTES/2014-12-25
- 2nd Data analysis was done by Dinh and Dr. Zhang Dinh/Dinh 2015/NOTES/2015-1-7 Kun:LabNotes/MONOD/2015-1-9
- 3rd Experiment was done by me (Kapa Hyper Lib. Prep + Seqcap-optimized) on AlanFung:LabNotes/2015/2015-1-15
- 3rd Data analysis was done by Dr. Zhang Kun:LabNotes/MONOD/2015-3-4
Summary
Experiment | Person | Capture Date | Lib Prep | Capture | Conclusion |
1 | Noi | 141104 | Tang | SeqCap | lower complexity and higher clonal rates due to Tang et assumption the DNA fragment ends |
2 | Noi | 141225 | Kapa Hyper | SeqCap | lower specificity and enrichment due to capture condition |
3 | Alan | 150115 | Kapa Hyper | SeqCap | Mapping rates, library complexity and capture specificity is high |
4 | Alan | 150714 | Swift | ? | ? |
Samples
- We only have 12 reactions from the swift methyl-seq kit and Dana needs to use it for her project so I can work on 4 samples.
- I am going to pick 1x tumor 2x plasma sample and 1x NC plasma
- I will pick the ones that have relatively low % trimmed (percentage of adapters being trimmed) and high % mapped rate from 2nd capture.
- I will pick the ones with high % on target and low % clonal from 3rd capture.
SAMPLE ID | Total PE reads | Total reads | Total reads after trimming | Total mapped reads | %trimmed | %mapped | |||
6T-2_map | 7,489,616 | 14,979,232 | 14,638,550 | 12,820,323 | 2% | 88% | |||
Sample | N_mapped_reads | N_non-clonal_reads | N_on-target_reads | N_non-clonal_on-target_reads | N_on-target_haplotypes | N_target_coverred | Pct_on-target | Pct_clonal | Enrichment_factor |
6P-3 | 28,124,349 | 27,174,711 | 20,126,384 | 19,455,168 | 7,299,019 | 48,605 | 71.60% | 3.40% | 254 |
PCP-3 | 9,225,527 | 9,067,481 | 6,226,370 | 6,133,490 | 1,984,048 | 47,803 | 67.50% | 1.70% | 210 |
NC-30 | 15,359,075 | 13,957,906 | 9,350,399 | 8,223,219 | 3,641,546 | 45,664 | 60.90% | 9.10% | 157 |
- After talking to Dr. Zhang we have decided to work on NC2, NC3, 6P1 and 7P8
Bisulfite Conversion using Zymo EZ DNA MEthylation-Lightning Kit (07/15/2015)
- Concentration based on Noi/NOTES/2014-10-22 Noi/NOTES/2014-11-30
Sample | Concentration ng/ul | 1ng Volume ul | 20ul H2O ul |
NC2 | 0.325 | 3.08 | 16.92 |
NC3 | 0.538 | 1.86 | 18.14 |
6P1 | 0.2 | 5.00 | 15.00 |
7P8 | 0.167 | 5.99 | 14.01 |
- 3.3 for 6p1
- 3.5 for 7p8
- Add 130ul lighting conversion reagent to 20ul of sample, compensate with water if less than 20ul.
- Vortex and centrifuge.
- (98C 8min > 54C 60min > 10C hold)
- Add 600ul of M-Binding Buffer to column with collection tube.
- Load the sample to column. Close the cap and mix by inverting the column several times.
- Centrifuge at full speed for 30s. Discard flow through.
- Add 100 μl of M-Wash Buffer to the column. Centrifuge at full speed for 30s.
- Add 200 μl of L-Desulphonation Buffer to the column and let stand at RT for 20 mins. After the incubation, centrifuge at full speed for 30 seconds.
- Add 200 μl of M-Wash Buffer to the column. Centrifuge at full speed for 30 seconds.
- Repeat this wash step.
- Place the column into a 1.5 ml microcentrifuge tube and add 10 μl of M-Elution Buffer directly to the column matrix. Centrifuge for 30 seconds at full speed to elute the DNA.
- The DNA is ready for immediate analysis or can be stored at or below -20°C for later use. -80C for long term storage.
Swift Methyl-Seq DNA Library Prep
Denature
- Transfer 15ul of bisulfite converted samples to 0.2ul PCR tube.
- Incubate at 95C for 2 mins
- Transfer to ice for 2 mins
Adaptase
- Prepare Master Mix
Content | 1x rxn ul | 4x rxn ul |
Low EDTA TE | 11.5 | 48.3 |
Buffer G1 | 4 | 16.8 |
Reagent G2 | 4 | 16.8 |
Reagent G3 | 2.5 | 10.5 |
Enzyme G4 | 1 | 4.2 |
Enzyme G5 | 1 | 4.2 |
Enzyme G6 | 1 | 4.2 |
Total | 25 | 105 |
- Add master mix to denatured samples vortex to mix and spin down
- (37C 15min > 95C 2min > 10C hold)
Extension
- Prepare Master Mix
Content | 1x rxn ul | 4x rxn ul |
Reagent Y1 | 2 | 8.4 |
Enzyme Y2 | 42 | 176.4 |
Total | 44 | 184.8 |
- Add 44ul of MM to each PCR tube
- Vortex spin down and run
- (98C 1min > 62 2min > 65C 5min > 4C Hold)
Post-Extension SPRI
- Vortex AMPure XP beads to resuspend.
- Add 101 μl resuspended AMPure XP beads beads to the ligation reaction.
- Mix well by pipetting up and down at least 10 times or vortex AMPure XP beads 10 seconds to resuspend.
- Incubate for 5 minutes at room temperature.
- Quickly spin the tube and place it on an appropriate magnetic stand to separate beads from supernatant.
- After the solution is clear (about 5 minutes), carefully remove and discard the supernatant.
- Be careful not to disturb the beads that contain DNA targets (Caution: do not discard beads).
- Add 200 μl of 80% freshly prepared ethanol to the tube while in the magnetic stand.
- Incubate at room temperature for 30 seconds, and then carefully remove and discard the supernatant.
- Repeat Step 5 once, for a total of two washes.
- Air the dry beads for 10 minutes while the tube is on the magnetic stand with the lid open.
- Elute the DNA target from the beads by adding 15 μl of 10 mM Tris-HCl, pH 8.0 or 0.1X TE.
- Note: Be sure not to transfer any beads. Trace amounts of bead carry over may affect the optimal performance of the polymerase used in the PCR master mix (2X) in the subsequent PCR step.
- Mix well by pipetting up and down, or on a vortex. After the solution is clear (about 5 minutes), carefully transfer 15 μl supernatant to a new PCR tube.
Ligation
- Prepare master mix
Content | 1x rxn ul | 4x rxn ul |
Buffer B1 | 3 | 12.6 |
Reagent B2 | 10 | 42 |
Enzyme B3 | 2 | 8.4 |
Low EDTA TE | 20 | 84 |
Total | 35 | 147 |
- 25C 15min > 4C Hold
Post-Ligation SPRI
- 1:1.2 elute with 20 ul Low EDTA TE.
Indexing PCR
- Add 2.5ul of indexing primer to directly to the sample.
Sample | Index D50X | Index D7XX |
NC2 | 502 | 701 |
NC3 | 502 | 702 |
6P1 | 502 | 703 |
7P8 | 502 | 704 |
- Prepare Master Mix
Content | 1x rxn ul | 4x rxn ul |
Low EDTA TE | 10 | 42 |
Buffer R1 | 10 | 42 |
Reagent R2 | 4 | 16.8 |
Enzyme R3 | 1 | 4.2 |
Total | 25 | 105 |
- Add 25ul MM to each indexed sample.
- Vortex and spin down.
- 98C 30sec > 10x (98C 10sec > 60C 30sec > 68C 60sec) > 4C Hold.
Post-PCR SPRI
- (1:0.8) 40ul beads elute with 20ul Low EDTA TE.