AlanFung:LabNotes/2015/2015-7-14: Difference between revisions

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* (98C 1min > 62 2min > 65C 5min > 4C Hold)
* (98C 1min > 62 2min > 65C 5min > 4C Hold)
==Post-Extension SPRI==
==Post-Extension SPRI==
* Vortex AMPure XP beads to resuspend.
* Add 83.5 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 20 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 18 μl supernatant to a new PCR tube.

Revision as of 23:10, 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)

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 83.5 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 20 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 18 μl supernatant to a new PCR tube.