Arichard:Notebook/midas140911: Difference between revisions

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==MagVigen DNA Select Focus 300/600 beads==
==MagVigen DNA Select Focus 300/600 beads==


* Motivation
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.


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


* 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?


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
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.
* 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===
===Samples===

Latest revision as of 21:34, 13 October 2014

MIDAS, September 11, 2014 (midas140911)[edit]

  • 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[edit]

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[edit]

File:Midas140911GelsSlide1.jpg File:Midas140911GelsSlide2.jpg File:Midas140911GelsSlide3.jpg

File:Midas140911GelsSlide4.jpg File:Midas140911GelsSlide5.jpg File:Midas140911GelsSlide6.jpg

Sequencing[edit]

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

MagVigen DNA Select Focus 300/600 beads[edit]

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[edit]

Gel result[edit]

File:.jpg

Sequencing[edit]