AlanFung:LabNotes/2015/2015-7-14: Difference between revisions
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* 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. | * 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. | * 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 | |||
{| class="wikitable" class="wikitable" class="wikitable" class="wikitable" class="wikitable" class="wikitable" | |||
|- style="font-size:12pt" align="center" valign="bottom" | |||
| width="71" height="15" | Content | |||
| width="65" | 1x rxn ul | |||
| width="65" | 4x rxn ul | |||
|- style="font-size:12pt" align="center" valign="bottom" | |||
| height="15" | Low EDTA TE | |||
| align="center" | 11.5 | |||
| align="center" | 48.3 | |||
|- style="font-size:12pt" align="center" valign="bottom" | |||
| height="15" | Buffer G1 | |||
| align="center" | 4 | |||
| align="center" | 16.8 | |||
|- style="font-size:12pt" align="center" valign="bottom" | |||
| height="15" | Reagent G2 | |||
| align="center" | 4 | |||
| align="center" | 16.8 | |||
|- style="font-size:12pt" align="center" valign="bottom" | |||
| height="15" | Reagent G3 | |||
| align="center" | 2.5 | |||
| align="center" | 10.5 | |||
|- style="font-size:12pt" align="center" valign="bottom" | |||
| height="15" | Enzyme G4 | |||
| align="center" | 1 | |||
| align="center" | 4.2 | |||
|- style="font-size:12pt" align="center" valign="bottom" | |||
| height="15" | Enzyme G5 | |||
| align="center" | 1 | |||
| align="center" | 4.2 | |||
|- style="font-size:12pt" align="center" valign="bottom" | |||
| height="15" | Enzyme G6 | |||
| align="center" | 1 | |||
| align="center" | 4.2 | |||
|- style="font-size:12pt" align="center" valign="bottom" | |||
| height="15" | Total | |||
| align="center" | 25 | |||
| align="center" | 105 | |||
|} | |||
* Add master mix to denatured samples vortex to mix and spin down | |||
* (37C 15min > 95C 2min > 10C hold) |
Revision as of 21:51, 15 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)