1000 20 Q17, with reads filtered only using the first four bases.
How we will get there
Quantify incorporation, cleavage, lead, lag, and decay based on fitted experimental results
Add images to monitor incorporation and cleavage completion
Run Mel’s code for lead, lag, and droop values
UV light coupling improvement
Increase cleave efficiency
Reduce dark base generation
Jon Schultz’s dark microbeads
Reduce stray light into bulk solution of sequencing reservoir impacting photobleaching, cat’s eye, and dark base generation
DISCS
Evaluate point of minimal DISCS + Pol complex interference with sequencing reaction
Lightning Terminators™
Test LTs with different dyes or non-dye LTs; Concentration
One pot buffers
Many sequencing runs across 2 instruments, automated data mining to aid in selecting quality predictors for non-cherry * picked clusters using 4 base key
Photoacid
Automated fast analysis for image pre-processing through signal processing
Current sequencing performance
Best run
Run ID
Library
Cycles
Mapped reads
Longest read length (bp)
>= 10 bp perfect reads
All reads Q score
Real filter Q score
S0592
cir16m
16
4927 (54% of all ROIs)
16
958 (19.4% mapped reads)
4903 10Q10.6
1859 10Q13.4
Analysis: lead and lag
Incomplete extension (ie) and carry forward (cf): for first 2, 3, 5 cycles fitting
S0592 (AG DISCS)
S0592
Q_score
ie_exp
cf_exp
dc_exp
Cluster_1268
CGTATCGACGATGCAG
30
0.21
0.05
0.13
Cluster_1268
CGTATCGACGATGCAG
30
0.16
0.13
0.01
Cluster_1268
CGTATCGACGATGCAG
30
0.2
0.13
0.01
Cluster_3718
ATGCAGTCGACGTACT
30
0.31
0.05
0.01
Cluster_3718
ATGCAGTCGACGTACT
30
0.36
0.05
0.105
Cluster_3718
ATGCAGTCGACGTACT
30
0.32
0.12
0.125
Cluster_5806
TCAGGACGATGACTGC
7.27
0.15
0.16
0.52
Cluster_5806
TCAGGACGATGACTGC
7.27
0.11
0.12
0.36
Cluster_5806
TCAGGACGATGACTGC
7.27
0.17
0.14
0.295
Cluster_65
GACTCTATTCTCAGTA
6.02
0.22
0.09
0.55
Cluster_65
GACTCTATTCTCAGTA
6.02
0.23
0.09
0.31
Cluster_65
GACTCTATTCTCAGTA
6.02
0.2
0.16
0.135
S0577 (No DISCS)
S0577
Q_score
ie_exp
cf_exp
dc_exp
Cluster_2532
CGTATCGACGATGCAG
12.04
0.06
0.05
0.01
Cluster_2532
CGTATCGACGATGCAG
12.04
0.18
0.06
0.025
Cluster_2532
CGTATCGACGATGCAG
12.04
0.18
0.05
0.13
Cluster_6083
ATGCAGTCGACGTACT
30
0.24
0.05
0.01
Cluster_6083
ATGCAGTCGACGTACT
30
0.32
0.05
0.01
Cluster_6083
ATGCAGTCGACGTACT
30
0.26
0.07
0.01
Cluster_10085
TCAGGACGATGACTGC
12.04
0.15
0.18
0.025
Cluster_10085
TCAGGACGATGACTGC
12.04
0.12
0.1
0.06
Cluster_10085
TCAGGACGATGACTGC
12.04
0.14
0.11
0.075
Cluster_19
GACTCTATTCTCAGTA
11.14
0.49
0.16
0.5
Cluster_19
GACTCTATTCTCAGTA
11.14
0.45
0.24
0.37
Cluster_19
GACTCTATTCTCAGTA
11.14
0.43
0.3
0.25
High decay in cycle 1 due to large signal loss in the 2nd cycle
High lag observed for “G” attributed to UV cleavage, with different bases displaying distinct features
The addition of DISCS results in a decrease in “cf” for G but an overall increase in “dc”
Generally, “ie” and “dc” perform worse than “cf,” with UV cleavage identified as the primary issue, followed by decay caused by DISCS, and darkbase generation ranking third