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
![](/blog/2024/01/08/january-8-update/sequencing-performance_hu72e6f0398377c9333b85dc0274506817_210303_1280x0_resize_catmullrom_3.png)
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 |
![](/blog/2024/01/08/january-8-update/bestrun-graphs_hu9c63c510f719bff3269807e25d19ab09_131014_1600x0_resize_catmullrom_3.png)
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
![](/blog/2024/01/08/january-8-update/mk302_hu58c875563244dba89b0815b3afb28f70_124474_0x640_resize_q75_catmullrom.jpg)
Mk 3.02
![](/blog/2024/01/08/january-8-update/mk303_huaf15ac99e28533a71c575ed46255fa62_81239_0x640_resize_q75_catmullrom.jpg)
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) |