Andrew:Notebook/AR150605 Tn5Titrate C1
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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
Reagents[edit]
File:AR150605 reagents-page-001.jpg
File:AR150605 reagents-page-002.jpg
Results[edit]
Gels[edit]
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.