Chrissy h. Roberts Room 246, Keppel St. DROPLET DIGITAL PCR NEXT GENERATION QUANTITATIVE PCR

Transcription

1 Chrissy h. Roberts Room 246, Keppel St. chrissyhroberts@yahoo.co.uk DROPLET DIGITAL PCR NEXT GENERATION QUANTITATIVE PCR

2 I WANT TO QUANTITATE AN INFECTIOUS LOAD IN A DNA SAMPLE CHLAMYDIA CHLAMYDIA CHLAMYDIA CHLAMYDIA

3 PCR IS A QUANTITATIVE PROCESS Baseline Exponential Plateau

4 THE TAKEOFF POINT OF THE EXPONENTIAL PHASE IS PROPORTIONAL TO THE STARTING NUMBER OF COPIES OF THE TEMPLATE MORE COPIES FEWER COPIES

5 QUANTITATIVE PCR DETERMINES THE AMOUNT OF DNA/RNA IN AN UNKNOWN SAMPLE BY COMPARISON TO A KNOWN REFERENCE 100 X 10

6 QUANTITATIVE PCR DETERMINES THE AMOUNT OF DNA/RNA IN AN UNKNOWN SAMPLE BY COMPARISON TO A KNOWN REFERENCE 100 ~50 10

7 THE REFERENCE DNA SAMPLE HAS A UNIFORM COMPOSITION AND IS A GOOD CLEAN TEMPLATE FOR DNA THINK OF IT LIKE PIET MONDRIAN S COMPOSITION WITH RED, YELLOW AND BLUE (1921)

8 BUT THE CLINICAL SAMPLE OF DNA IS REALLY AN HIGHLY COMPLEX MICROSCOPIC UNIVERSE IN WHICH THOUSANDS OF CHEMICALS AND MILLIONS OF DNA MOLECULES FROM MANY SPECIES ARE INTERACTING. INDIVIDUAL SAMPLES DIFFER WIDELY! MORE LIKE MONDRIAN S BROADWAY BOOGIE WOOGIE ( )

9 IT HAS ALWAYS BEEN ACCEPTED THAT Q-PCR PERFORMS WITH THE SA EFFICIENCY WHEN SUPPLIED WITH DNA SAMPLES OF DIFFERENT COMPOSITION = I.E. THAT THE STANDARDS ACCURATELY PREDICT THE CLINICAL SAMPLES BECAUSE IF THAT WEREN T TRUE THEN IT WOULD ALL BE A LOAD OF RUBBISH

10 BECAUSE IT WOULD AN THAT THIS HAPPENS... [OBSERVED] 100 [TRUE] 10

11 OF COURSE IT IS A LOAD OF RUBBISH PCR EFFICIENCY IS AFFECTED BY SAMPLE COMPOSITION AND ALSO BY THE CONCENTRATION OF THE TEMPLATE IN THE SAMPLE THIS MAKES Q- PCR INACCURATE A LOT OF THE TI

12 CAN T WE JUST COUNT THEM? AS IN ACTUALLY COUNT THEM! 1 CHLAMYDIA 2 3 CHLAMYDIA CHLAMYDIA 4 CHLAMYDIA AS IN, WITHOUT HAVING TO REFER TO A CALIBRATOR?

13 IMAGINE A GA IN WHICH THERE ARE A LOAD OF BOXES AND FIVE DUCKS YOU THROW THE DUCKS IN THE AIR AND THEY FALL IN TO THE BOXES

14 IT S VERY UNLIKELY THAT YOU LL GET TWO DUCKS TO LAND IN THE SA BOX! BECAUSE THERE ARE LOTS OF BOXES AND THERE ARE NOT A LOT OF DUCKS AND IF YOU COUNT UP THE NUMBER OF BOXES THAT HAVE A DUCK IN THEM, IT IS THE SA AS THE NUMBER OF DUCKS THAT YOU HAD TO BEGIN WITH.

15 DO THE SA THING WITH DNA INSTEAD OF DUCKS, AND DROPLETS OF PCR MIX INSTEAD OF BOXES AND YOU GET DIGITAL PCR

16 EACH DROPLET GETS ZERO OR ONE TEMPLATE MOLECULE (0 1 COPY OF CHLAMYDIA DNA) Because there are lots of droplets and not lots of templates most droplets get zero templates

17 AFTER PCR most droplets have no PCR product Some have a detectable PCR product those are the ones that had a template in them Count them up, just like the boxes that had ducks in them!

18 DROPLETS ARE READ IN SINGLE FILE ON A MODIFIED FLOW CYTOTER WITH OIL BASED FLUIDICS IS THERE ANY PCR PRODUCT IN THIS DROPLET? YES / NO ANSWER (THAT S DIGITAL)

19 Chlamydia DNA positive droplets fluoresce on the FAM channel (green in figure) 5000 P_022_A OMCB 4000 Human DNA positive droplets fluoresce on HEX channel (red) PLASMID Count up the positives and negatives

20 OK, SO IT IS A BIT MORE COMPLICATED THAN THAT... but you can read about that for yourselves in the following paper. Hindson, B. J et. al High-throughput droplet digital PCR system for absolute quantitation of DNA copy number. Analytical chemistry 83:

21 Imagine the dpcr array to be a random sampling of an infinite universe of chambers filled with the DNA sample. The true mean concentration of the template in the sample (lambda) can be estimated using sample estimators of the probability (p) of any droplet containing a template molecule. p can be estimated as p-hat = H/C where H is the number of observed hits (PCR positive droplets) and C is the number of droplets. The sample distribution is normal mean = p sd = sqrt(p((1-p)/c)))

22 The concentration/droplet and 95% confidence interval is estimated as PROBABILITY OF ANY DROPLET BEING POSITIVE p_hat = H/C (# hits/ # droplets) CONCENTRATION PER DROPLET (lambda_hat) lambda_hat = -ln(1- p_hat) #true as long as the droplet count is very high CONFIDENCE INTERVAL of P_HAT p_hat[low,high] = p_hat ± 1.96*sqrt(p_hat((1-p_hat)/C)) CONFIDENCE INTERVAL OF LAMBDA_HAT lambda_hat[low] = -ln (1 - p_hat[low]) lambda_hat[high] = -ln (1 - p_hat[high])

23 lambda_hat (copies per droplet) * number of droplets = concentration (copies per reaction) copies per reaction / (droplets * average droplet volume [0.91 e-9])*1,000,000) = Copies per microlitre copies per microlitre * dilution factor * volume of total swab extract CONCENTRATION PER SWAB =