Dinh 2011/NOTES/2011-8-1

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Barcoding BSPP probes[edit]

0. Oligos re-ordered[edit]

Taqα1_guide_bot:
5'-CAGGCAGATGTTATCGAGGTCCGAC -3' (Tm ~ 68C)

Taqα1_insert_guide_bot:
5'-GTCGGACCTCGTGAGAGTGGTGTANNNNNNNGATCTCGGTGGTCGCCGTATCATTATAACATCTGCCTG - 3' (Tm ~ 81C)
Insert = 44 residues
APV6_Splint:
5'-TACACACTACCCGTGGGGTCATATCGGTCA-3' (Tm ~ 73C)


1. Production of probes[edit]

  • Make master mix of all 1st round amplicon probes subset with multiplex E ratios (see Noi's notes)
SUBSET             Vol(ul)
S1 (diluted to 0.1x) 0.33
S2 (diluted to 0.1x) 0.48
S3                   1.32
S4                   5.67
S5                  27.39
Exp1                 1.33
Exp2                 1.05
Exp3                 1.51
-------------------------
  • PCR reaction of probes with phosphorylated primer at common flanking sequence
Production PCR:                    1x     32x
10nM 1st round amplicon          0.2ul   6.4ul
2x Kapa SYBR qPCR MM              50ul  1600ul
100uM pAP1V61U                   0.4ul  12.8ul
100uM AP2V6                      0.4ul  12.8ul
H2O                               49ul  1568ul
----------------------------------------------
                                 100ul x 32
----------------------------------------------
95C 30s -> (95C 3s -> 55C 30s -> 60C 20s) x 14 cycles -> 60C 2min -> 15C hold
Ethanol precipitation, re-purified with Qiaquick columns elute with EB.
Yield (Nanodrop): 74ng/ul * 120 ul + 78ng/ul * 120 = 18.240 ug

2. Circularization with circle-helper and Ampligase[edit]

  • Ampligase to anneal 5' to 3' ends with APV6_Splint (1:1 ratio)
Circularization reaction:
1 ug of dsDNA probes (150 nt, MW = 91,268 g/moles) 
Probes (1.09x10^-11 moles in 1 ug)
APV6_Splint (1.09x10^-11 moles = 100x10^-12 moles/ul*0.109ul)
Splint : Use 0.109ul 100uM APV6_Splint per 1 ug of probes (1:1 ratio). 
Ampligase (Epicentre) : Use 1 ul of Ampligase  
Ampligase buffer : Use 1x
Reaction:
1 ug amplified probes (74ng/ul)   13.5 ul
APV6_Splint (5uM)                  2.2 ul
      dilute APV6_Splint (5 ul + 95 ul ddH20)
Ampligase                          1.0 ul
Ampligase buffer                   2.0 ul
ddH2O                              1.3 ul
-----------------------------------------
                                  20.0 ul total
Program:
95C 30S -> 94.8C for 30s, -0.2C per cycle x 148 cycles -> (65C 10min -> 94C 30s)x10 -> 65C forever -> 37C 30 s -> Add 1 ul ExoI and ExoIII each -> 37C 45 min -> 4C hold
  • Exonuclease I/III to remove all linear DNA
Purified with columns, elute with 30ul EB.
Nanodrop: 1.6ng/ul, 2.43 OD

Repeat circularization due to low yield

Reaction:
1 ug amplified probes (74ng/ul)   13.7 ul
APV6_Splint (5uM)                  3.3 ul
     3:2 ratio
     Final conc = 825nM => Tm = 73.14C 
Ampligase                          1.0 ul
Ampligase buffer                   2.0 ul
-----------------------------------------
                                  20.0 ul total
Program A and B:
95C 30S -> [94.8C for 30s, -0.2C per cycle x 148 cycles -> 65C hold -> add 1ul Ampligase]x accidentally cycled here for total 2x ->(65C 10min -> 94C 30s)x11 -> 65C forever -> 37C 1 min -> Add 1 ul ExoI and ExoIII each -> 37C 45 min -> 90C 10 min 4C hold
Program C:
95C 30S -> 94.8C for 30s, -0.2C per cycle x 148 cycles -> 65C hold -> add 1ul Ampligase ->(65C 10min -> 94C 30s)x11 -> 65C forever -> 37C 1 min -> Add 1 ul ExoI and ExoIII each -> 37C 45 min -> 90C 10 min-> 4C hold
Program D:
(94C 30s -> 65C 2 min)x40 -> 65C forever -> 37C 1 min -> Add 1 ul ExoI and ExoIII each -> 37C 1 hr -> 90C 10 min -> 4C hold
  • Quantify amount of circularized probes using TBU gel
File:ZhangLab 2 2011-08-10 DMR330K-E circularization.png
A - Low mass ladder (0.6ul)
B - Ligation product from program C (can see the circularized probes band)
C - Ligation product from program D (incubation with Ampligase at high temperature have exonucleas activity ?)
D - Linear dsDNA probes
E - 10 bp ladder

  • Adding ampligase after gradual lowering of temperature from 95C helps, allows for longer incubation at 65C?
  • Try titrating the amount of APV6_Splint to be added

Repeat circularization with titrating splint amount

Reaction:
1 ug amplified probes (78ng/ul)   12.8 ul
APV6_Splint (1uM) 
     probes to splint               
     1:1   =   11 ul
     1:2   =    ul
     1:4   =   22 ul
Ampligase buffer                   2.7 ul
H2O
     1:1   =   0.5 ul
     1:2   =   6.0 ul
     1:4   =  8.75 ul
-----------------------------------------
                                  27.0 ul total
Program: 
95C 30S -> 94.8C for 30s, -0.2C per cycle x 199 cycles -> 55C hold -> add 1ul Ampligase @ 20 min -> 55C 2 hr (remove 5ul for gel) -> 37C 1 min -> Add 1 ul ExoI/ExoIII mix -> 37C 1 hr -> 90C 10 min-> 4C hold
File:TitratingCircularization.jpg
Mix products from all tubes. Purified with 1.8x AMPure beads, elute with water.
Nanodrop: 1.6ng/ul

3. Annealing of guide oligo and digestion[edit]

  • Quantify amount of circularized ssDNA probes using gel
  • Use 5:1 ratios of guide oligos
Circularized probes:
1 ug of ssDNA circular probes (150 nt, MW = 45,634 g/moles) = 2.191x10^-11 moles
TaqαI_Guide: 
5 * 2.191x10^-11 moles / 100x10^-12 moles/ul = 1.0955 ul
-Use 1.1 ul 100uM TaqαI guide oligo per 1 ug of circle ssDNA probes
TaqαI : 
1 unit cuts 121 sites in 1 ug Lambda DNA (48,502bp, MW = 2.946x10^7g/mol) in 1 hour at 65C
Unit per sites*moles = 1 unit * 121^-1 sites^-1 * 2.946x10^13 mol^-1 = 2.946x10^11 
-Use 2.946x10^-11 sites*moles x2.191x10^-11 = 3.523 units per 1 ug of circle ssDNA probes
Reaction:
20 ul Circled probes
0.64 ul TaqαI_Guide (0.5uM)
1 ul TaqαI (20U/ul)
4 ul NEB Buffer 4 (10x)
0.4 ul BSA (100x)
---------------------
40ul total
Program:
65C 20min -> (Add enzyme) 65C for 1 hr -> 80C 20 min -> 4C hold

Purify with 1 column, elute in water
Nanodrop: too low
Gel:
File:Taq1digest.jpg

4. Annealing of guide oligo, fill in with Stoffel, and Ampligase[edit]

  • Set up as in capture reaction but with higher annealing temperature
  • Use 2:1 ratio of guide oligos
TaqαI digested ssDNA probes : 1ug
1 ug of ssDNA probes (150 nt, MW = 45,634 g/moles) = 2.191x10^-11 moles
TaqαI_RandBar_guide : 
- Use 0.109 ul of 100uM Insert_guide oligo per 1 ug of digested probes

Stoffel (10U/ul): One unit per 10nmoles dNTPs to incorporate per 30 min in 10 min incubation at 74C
0.0219nmolesx44nt =  0.9636nmoles dNTPs to incorporate
Reaction will cycle to 74C for a total of 30 min
Assumes One unit per 40nmoles, thus ~0.5 Units Stoffel per 1 ug of digested ssDNA probes.

dNTP (10mM = 10nmoles/ul): Optimal is 10x the amount required for 100% of targets. 
0.0219nmolesx44nt =  0.9636 nmoles dNTPs to incorporate
100*0.9636nmoles/10nmoles/ul = 9.636ul
- For 100:1 guide to probes ratio, use 10 ul of 10mM dNTP per 1 ug of digested ssDNA probes
Ampligase (5U/ul): One unit per 50% of cos site of 1 ug lambda DNA in 1 minute at 45C
- Use 1 unit

Ampligase buffer (10x): Use 1x
Reaction Mix:
SLN for ~ 5 ng of digested product
Use 0.5nmoles of dNTP = (1nmoles/ul)x 0.5 ul
0.5 ul dNTP (1 mM)
1.0 ul Stoffel (10U/ul)
0.5 ul Ampligase (5U/ul)
0.5 ul Ampligase buffer (10x)
2.5 ul H2O 
---------------------------
5.0 ul total

Program:
Program C:
95C 30S -> 94.8C for 30s, -0.2C per cycle x 184 cycles -> 58C hold -> add SLN ->(58C 10min -> 94C 30s)x11 -> 58C forever -> 37C 1 min -> Add 1 ul ExoI and ExoIII each -> 37C 45 min -> 90C 10 min-> 4C hold

5. PCR Amplification of circled probes with common sequence primers[edit]

  • PCR reaction of probes with primer at common flanking sequence
Production PCR:                    1x     32x
circled barcoded probes            ul   ul
2x Kapa SYBR qPCR MM               ul   ul
100uM pAP1V61U                     ul   ul
100uM AP2V6                        ul   ul
H2O                                ul   ul
----------------------------------------------
                                 100ul x 32
----------------------------------------------
95C 30s -> (95C 3s -> 55C 30s -> 60C 20s) x 15 cycles -> 60C 2min -> 15C hold
Ethanol precipitation, re-purified with Qiaquick columns elute with EB.
Yield (Nanodrop):

6. Probes preparation - adapters removal[edit]

7. Capture using barcoded probes[edit]

8. Amplification with AmpR6.3IndXX and AmpF6.4 primers[edit]

9. Set up new recipe on GAIIx[edit]

  • Run sequencing to sequence 4 reads using 4 set of reading primers