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

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)

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)