Andrew:Notebook/AR150605 Tn5Titrate C1

From ZhangLabWiki
Revision as of 15:11, 12 June 2015 by >Andrew (→‎Gels)
(diff) ← Older revision | Latest revision (diff) | Newer revision → (diff)
Jump to navigation Jump to search

Tn5 titration on the C1 using GM12878[edit]

  • This is essentially a repeat of AR150602, in which the mouse DNA spike-in was not diluted

Motivation

  • Library complexity depends on the ratio of Tn5 to template.
  • Tn5-059 concentration is normalized to EZ-Tn5. The current batch of Tn5-059 is 25X = 17.5 uM (1X = 700 nM).
  • Illumina uses 40 nM Tn5-59 to 5 ng purified DNA, which is 400 fmole/ng assuming a 50 uL reaction
  • I have been using 0.1X = 70 nM to 6 pg for a single cell, which is 368 fmole/ng in a 31.5 nL volume
  • Each fragment requires adapters on both ends; half will be orange/orange or blue/blue, which are non-functional.
  • A million unique reads will therefore consume 4 million molecules of Tn5
  • 70 nM in 31.5 nL is approximately 1.33 billion molecules

Goal

  • Compare a range of Tn5/template ratios for Tn5-059 supermutant versus EZ-Tn5
  • Perform library prep directly on single cells
  • Lyse with hypotonic condition and accessibilize DNA with Qiagen Protease
  • Evaluate conditions based on library complexity and coverage bias

Protocol[edit]

File:Protocol Worksheet AR150527 Tn5 titration v1-page-001.jpg

File:Protocol Worksheet AR150527 Tn5 titration v1-page-002.jpg

File:Protocol Worksheet AR150527 Tn5 titration v1-page-003.jpg

File:TempProfile.png

Reagents[edit]

File:AR150605 reagents-page-001.jpg

File:AR150605 reagents-page-002.jpg

Results[edit]

Gels[edit]

File:AR150605 qPCR.png

File:AR150605 gelsSlide1.jpg

File:AR150605 gelsSlide2.jpg

Sequencing[edit]

  • Both fractions, 100-200 and 200-800 bp, were quantified by Qubit and qPCR. The 100-200 bp fraction had ~16 times higher concentration than the 200-800 bp, which corresponds to the gel intensity.
  • This indicates that the P5 and P7 regions are intact, although the smaller size fraction is too small to have actual inserts.
  • The 200-800 bp fraction was run on a MiSeq. The flow cell clustered at 935k/mm^2, but there were no clusters that passed filter.
  • The cycle intensities were extremely erratic.
  • If the machine was able to quantify cluster density, then the first base must have been incorporated.
  • It does not make sense why the sequencing would have failed.

Follow-up[edit]

  • It is possible that the presence of the short transposon adapter sequences in the harvested amplicons are leading to off-target products during the off-chip 2nd PCR.
  • Cleaning up the harvested amplicons before running off-chip 2nd PCR could address this issue.
  • I selected 8 single-cell libraries and did Ampure XP bead purification, then repeated the off-chip PCR.