Daniel:Notebook/ComboLock/2017-5-16: Difference between revisions

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===UMI Counting===
===UMI Counting===


<gallery perrow=2 heights=300px widths=300px caption="UMI Count histograms ">
<gallery perrow=3 heights=300px widths=300px caption="UMI Count histograms ">
File:Sample.neb-histogram.png|UMI count histogram from the NEB sample
File:Sample.neb-histogram.png|UMI count histogram from the NEB sample
File:Sample.tf-histogram.png|UMI count histogram from the ThermoFisher sample
File:Sample.tf-histogram.png|UMI count histogram from the ThermoFisher sample
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File:Poscon.tf-basecomplexity.png|Positive Control (TF) base complexity
File:Poscon.tf-basecomplexity.png|Positive Control (TF) base complexity
File:RandomUMIs-basecomplexity.png|Randomly generative UMI base complexity
File:RandomUMIs-basecomplexity.png|Randomly generative UMI base complexity
</gallery>
===Knee Plots (5/19/2017)===
Added from analysis performed yesterday (5/18). The knee plots show the UMIs, ordered by number of reads (descending), vs. total fraction of reads. Only the random UMI samples go up to 1 on the y axis, but that's because the others don't have 100% alignment.
<gallery perrow=3 heights=300px widths=300px caption="Knee Plots for MiSeq 20170508-Fraction of UMIs vs Fraction of Reads">
File:Sample.neb-kneeplot.png|Sample (NEB)
File:Sample.tf-kneeplot.png|Sample (TF)
File:Poscon.neb-kneeplot.png|Positive control (NEB)
File:Poscon.tf-kneeplot.png|Positive control (TF)
File:Dephos.tf-kneeplot.png|Dephosphorylated sample (TF)
File:RandomUMI.250K-kneeplot.png|Random UMI
</gallery>
</gallery>


===Discussion===
===Discussion===


Sequencing results are good overall, at least good enough to move on to the BSA positive control and possibly cells for mRNA.
Sequencing results are good overall, at least good enough to move on to the BSA positive control and possibly cells for mRNA. Couple of notes:
 
*Production run positive control yielded correct sequences
*Samples had additional empty UMIs to theoretical Poissonian zero
*Dephosphorylated had most (72%) empty UMIs
*ThermoFisher had slightly better UMI complexity
*The knee plots show that the dephosphorylated sample had the highest AUC, which is actually bad for this type of analysis
 


[[Category:ComboLock]] [[Category:20170428]]
[[Category:ComboLock]] [[Category:20170428]]

Latest revision as of 15:33, 19 May 2017

Production Run (Started Friday 4/28; Libary Prep)[edit]

Back to Calendar

Sequencing Results[edit]

These results are from the sequencing run performed 5/8/2017.

Read Statistics[edit]

Sample Condition Reads Aligned Reads Unaligned Pct Aligned Missing UMIs (%) Poisson Lambda Poissonian E[0] (%) UMI Missing Enrichment
1X NEB Sample 254728 21861 92.1% 34.17 3.89 2.0 16.7
5AX NEB Positive Control 119220 10206 92.1% 31.2 1.82 16.2 1.9
1Y TF Sample 197959 23195 89.5% 42.53 3.02 4.9 8.7
5AY TF Positive Control 63074 6955 90.1% 51.37 0.96 38.3 1.3
4AY TF Dephosphorylated 133660 15412 89.7% 72.47 2.03 13.1 5.5
NA Randomly Generated 250,000 UMIs     2.26 3.81 2.21 1.0

UMI Counting[edit]

Base Complexities[edit]

Knee Plots (5/19/2017)[edit]

Added from analysis performed yesterday (5/18). The knee plots show the UMIs, ordered by number of reads (descending), vs. total fraction of reads. Only the random UMI samples go up to 1 on the y axis, but that's because the others don't have 100% alignment.

Discussion[edit]

Sequencing results are good overall, at least good enough to move on to the BSA positive control and possibly cells for mRNA. Couple of notes:

  • Production run positive control yielded correct sequences
  • Samples had additional empty UMIs to theoretical Poissonian zero
  • Dephosphorylated had most (72%) empty UMIs
  • ThermoFisher had slightly better UMI complexity
  • The knee plots show that the dephosphorylated sample had the highest AUC, which is actually bad for this type of analysis