Arichard:Notebook/midas140911: Difference between revisions

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Revision as of 21:33, 13 October 2014

MIDAS, September 11, 2014 (midas140911)

  • Gwen asked for a run with only cerebellum, all of which is NeuN +, with both AD patient 25-00 and control patient 7-03 in the same run.
  • Based on positive results from the last run (midas140828), I removed the buffer headspace from all microwell arrays before adding oil overlay. This was an effort to prevent runaway reactions.
  • Also based on positive results from the last run, I skipped ALS/NS/PolI and EtOH precipitation before Nextera. This made the run much faster.

Samples

Gel lane Sample type Array#Pos# Index SeqID (If selected)
1 NeuN+ Cbl from 7-03 sorted May 20, 2014 a1p1 16 AR56-PL703May20-Sep11-16
2 NeuN+ Cbl from 7-03 sorted May 20, 2014 a1p1 25 AR57-PL703May20-Sep11-25
3 NeuN+ Cbl from 7-03 sorted May 20, 2014 a2p3 26 N/A
4 NeuN+ Cbl from 7-03 sorted May 20, 2014 a2p3 27 N/A
5 NeuN+ Cbl from 7-03 sorted May 20, 2014 a2p4 28 AR58-PL703May20-Sep11-28
6 NeuN+ Cbl from 7-03 sorted May 20, 2014 a3p7 29 AR59-PL703May20-Sep11-29
7 NeuN+ Cbl from 7-03 sorted May 20, 2014 a3p9 30 N/A
8 NeuN+ Cbl from 7-03 sorted May 20, 2014 a3p10 31 AR60-PL703May20-Sep11-31
9 NeuN+ Cbl from 7-03 sorted May 20, 2014 a4p11 33 N/A
10 NeuN+ Cbl from 7-03 sorted May 20, 2014 a4p12 34 N/A
11 NeuN+ Cbl from 7-03 sorted May 20, 2014 a4p12 35 N/A
12 NTC from+B51 array loaded with 7-03 a4p12ntc 36 N/A
13 NeuN+ Cbl from 25-00 sorted May 20, 2014 a5p13 37 AR61-PL2500May20-Sep11-37
14 NeuN+ Cbl from 25-00 sorted May 20, 2014 a5p13 38 AR62-PL2500May20-Sep11-38
15 NeuN+ Cbl from 25-00 sorted May 20, 2014 a6p14 39 N/A
16 NeuN+ Cbl from 25-00 sorted May 20, 2014 a6p14 40 N/A
17 NeuN+ Cbl from 25-00 sorted May 20, 2014 a6p15 41 N/A
18 NeuN+ Cbl from 25-00 sorted May 20, 2014 a7p17 42 AR63-PL2500May20-Sep11-42
19 NeuN+ Cbl from 25-00 sorted May 20, 2014 a7p18 43 AR64-PL2500May20-Sep11-43
20 NeuN+ Cbl from 25-00 sorted May 20, 2014 a8p20 44 N/A
21 NeuN+ Cbl from 25-00 sorted May 20, 2014 a8p20 45 N/A
22 NeuN+ Cbl from 25-00 sorted May 20, 2014 a8p21 46 N/A
23 NeuN+ Cbl from 25-00 sorted May 20, 2014 a8p22 47 N/A
24 NTC from array loaded with 25-00 a8p21ntc 48 N/A

Size select gels after Nextera

File:Midas140911GelsSlide1.jpg File:Midas140911GelsSlide2.jpg File:Midas140911GelsSlide3.jpg File:Midas140911GelsSlide4.jpg File:Midas140911GelsSlide5.jpg File:Midas140911GelsSlide6.jpg

Sequencing

  • These libraries were submitted for sequencing in HL173. They were not screened.

MagVigen DNA Select Focus 300/600 beads

Motivation

  • PA gel size selection is time and labor intensive. NVigen makes a kit called MagVigen DNA select 300/600 (Cat #Kr61001-300/600) for NGS library prep. This would save ~0.5 to 1 day on each run.

Goal

  • Answer each of the following questions:
    • What is the loading capacity of the beads?
    • How clean is the selection?
    • Will the library cluster as densely?
    • Will the pass filter rate suffer?
  • Run side-by-side gel comparison of bead vs. PA gel size selection and send the gel images to Aihua at NVigen as per our agreement.

Strategy

  • Save 5 out of 20 ul from each of 9 libraries (only high-concentration libraries as assessed by PA gel) to pool and run on MagVigen beads. I carried on with size selection for the remaining 15 ul of each of the 9 positive libraries.

Samples

Gel result

File:.jpg

Sequencing