9:30a – 12:40a
Goals Today
- Start Bowtie analysis of Deep-Seq data
- Apply new drift correction approach to STORM data, compare spot quality
- STORM imaging of PH clusters + DNA FISH
- if anti-PH fails, try to restain with anti-Flag
- nuclear signal is pretty weak in PH. staining PH-WT-FLAG cells with anti-flag.
Deep sequencing analysis
- built indexed lib fasta files
- Ran Bowtie
- built matlab bioindexed files
- converted to matlab fast-indexed structures
More Bead alignment
- cp2tform (control point to tform) doesn’t do rotation translation without forcing scaling option
- I made a clone function of this version which accepts ‘rotation translation’ as an input:
WarpPoints.m(in TSTORM/Code/Functions on TUCK) - Found more issues with FeducialDriftCorrection – cross correlation fails.
crosslinked beads
- 12/05/13 has crosslinked beads — see if these stay put better
- definetely better stuck. (should quantify);
Drift uncertainties for E1cell2 (unlinked beads)
Distance in nm between matched beads after the warp. sorted in rank order.
implemented using best pair alignment.
47.7950 48.3579 56.3873 60.3818 66.6519
2.4474 4.1889 5.8574 7.7761 8.2402
1.0944 3.7067 28.7937 34.8875 36.6357
0.0742 0.0886 12.5635 15.0466 19.3501
11.5580 11.8013 13.9580 15.5071 17.5983
9.3785 20.3346 22.2668 26.5540 27.4070
4.8066 5.5000 36.0185 40.2484 42.5206
1.0492 5.2735 10.4605 16.4393 52.9124
23.1428 27.3867 68.5654 88.9050 402.9921
35.1398 41.7546 58.7860 64.8746 70.3163
1.4286 5.0899 15.3220 18.8834 24.4787
0.3453 1.2389 12.9271 19.2681 28.1222
8.3024 11.3135 13.4250 31.0762 34.3580
3.6465 13.4411 42.6611 49.2972 52.0924
Typical uncertainties in alignment of 12/13 beads crosslinked to coverslip. (in nm)
0.8811 0.9095 0.9095 0.9095 0.9095
2.5366 3.5464 9.6424 10.3101 11.0085
0.1880 0.3737 1.2259 3.0643 4.8915
0.2896 0.3411 2.6533 2.7363 2.7930
0.1344 0.2061 1.2882 1.6983 1.8770
0.0060 0.0085 0.9791 1.1393 1.7360
0.5859 0.7642 0.8509 1.0569 1.8491
2.4244 3.6952 6.0305 13.0327 13.1788