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=Production Run (Started [[Daniel:Notebook/ComboLock/2017-4-28|Friday 4/28]]; [[Daniel:Notebook/ComboLock/2017-5-3|Libary Prep]])= [[Daniel:Notebook/ComboLock|Back to Calendar]] ==Sequencing Results== These results are from the sequencing run performed 5/8/2017. ===Read Statistics=== {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#66CCFF;font-size:18pt;font-weight:bold" align="center" | width="150" height="73" | Sample | width="220" | Condition | width="150" | Reads Aligned | width="120" | Reads Unaligned | width="120" | Pct Aligned | width="120" | Missing UMIs (%) | width="120" | Poisson Lambda | width="155" | Poissonian E[0] (%) | width="125" | UMI Missing Enrichment |- style="font-size:18pt" align="center" valign="bottom" | height="23" | 1X | NEB Sample | align="center" | 254728 | align="center" | 21861 | align="center" | 92.1% | align="center" | 34.17 | align="center" | 3.89 | align="center" | 2.0 |style="color:#FF0000" align="center" | 16.7 |- style="background-color:#BFBFBF;font-size:18pt" align="center" valign="bottom" | height="23" | 5AX | NEB Positive Control | align="center" | 119220 | align="center" | 10206 | align="center" | 92.1% | align="center" | 31.2 | align="center" | 1.82 | align="center" | 16.2 |style="font-weight:bold" align="center" | 1.9 |- style="font-size:18pt" align="center" valign="bottom" | height="23" | 1Y | TF Sample | align="center" | 197959 | align="center" | 23195 | align="center" | 89.5% | align="center" | 42.53 | align="center" | 3.02 | align="center" | 4.9 | align="center" | 8.7 |- style="background-color:#BFBFBF;font-size:18pt" align="center" valign="bottom" | height="23" | 5AY | TF Positive Control | align="center" | 63074 | align="center" | 6955 | align="center" | 90.1% | align="center" | 51.37 | align="center" | 0.96 | align="center" | 38.3 |style="font-weight:bold" align="center" | 1.3 |- style="font-size:18pt" align="center" valign="bottom" | height="23" | 4AY | TF Dephosphorylated | align="center" | 133660 | align="center" | 15412 | align="center" | 89.7% | align="center" | 72.47 | align="center" | 2.03 | align="center" | 13.1 | align="center" | 5.5 |- style="background-color:#BFBFBF;font-size:18pt" align="center" | height="46" | NA | Randomly Generated 250,000 UMIs | align="center" | | align="center" | | align="center" | | align="center" | 2.26 | align="center" | 3.81 | align="center" | 2.21 |style="font-weight:bold" align="center" | 1.0 |} ===UMI Counting=== <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.tf-histogram.png|UMI count histogram from the ThermoFisher sample File:Poscon.neb-histogram.png|UMI count histogram for positive control samples, from NEB File:Poscon.tf-histogram.png|UMI count histogram for positive control samples, from TF File:Dephos.tf-histogram.png|UMI count histogram for dephosphorylated samples (TF) File:RandomUMI.250K-histogram.png|UMI count histogram for randomly generated UMI list </gallery> ===Base Complexities=== <gallery perrow=3 heights=300px widths=300px> File:Sample.tf-basecomplexity.png|Sample (TF) UMI base complexity File:Sample.neb-basecomplexity.png|Sample (NEB) UMI base complexity File:Poscon.tf-basecomplexity.png|Positive Control (TF) 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> ===Discussion=== 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]]
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