January 8 update

Proposed January 8 goal

Proposed January 8 goal

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

Characterization of imaging systems

Mk 3.02

Mk 3.03

Specs MK3.02 MK3.03
Magnification 5.71x 4.04x
CMOS Type SONY IMX477, 1.55um/pixel (*mono$700) SONY IMX183 2.4um/pixel ($550)
CMOS to Chip Distance 210.1mm 166.9mm
CMOS size 6.28x4.71mm (4056x3040 pixel) 13x8.76mm (5440x3648 pixel)
FOV (in mm2) 1.1x0.83mm 3.23x2.17mm (7.7x larger Img area)
Imaging Lens 5x Olympus NA=0.15 ($500) 5x Bolioptics NA=0.13 ($50)