Sequencing in situ by multiplexed padlock probes and rolling circle amplification (RCA)

Daniel Fürth
Meletis Lab

DMC lab meeting
9th May 2016

daniel.furth@ki.se

Padlock probes

b, b' : 20nt target sequences.

a, a' : complementary sequence.

c: detection sequence

 

 linear, oligonucleotide single stranded construct construct

Nilsson et al. (1994) Science

Padlock probes

b, b' : 20nt target sequences.

a, a' : complementary sequence.

c: detection sequence

 

 

 linear, oligonucleotide single stranded construct construct

5'

If last nt of the 3’ arm are non-complementary to the target sequence:

     ligation is comprimised (padlock is not locked)

3'

5'

3'

Nilsson et al. (1994) Science

Rolling Circle Amplification (RCA)

If ligation is not comprimised padlock probe will not be washed away.

Phi29 DNA Polymerase yields 800-1000 copies of a padlock sequence per hour.

Rolling Circle Amplification (RCA)

If ligation is not comprimised padlock probe will not be washed away.

Phi29 DNA Polymerase yields 800-1000 copies of a padlock sequence per hour.

Rolling Circle Amplification (RCA)

If ligation is not comprimised padlock probe will not be washed away.

Phi29 DNA Polymerase yields 800-1000 copies of a padlock sequence per hour.

Multiplexing

Reverse transcription

RNase H digestion

Hybridization

Ligation

Rolling Circle Amplification

FISH

Multiplexed padlock probes

Mats Nilsson and Jens Hjerling-Leffler

Multiplexed padlock probes

Mats Nilsson and Jens Hjerling-Leffler

Multiplexed padlock probes

Mats Nilsson

Multiplexed padlock probes

Mats Nilsson

Thank you!

Concurrency and parallel programming

  • Multi-threaded applications through             .
#include <string>
#include <iostream>
#include <thread>

using namespace std;

//The functions we want to make the thread run.
void task1(string msg)
{
    cout << "task1 says: " << msg;
}

void task2(string msg)
{
    cout << "task1 says: " << msg;
}

//Main loop.
int main()
{
    thread t1(task1, "Task 1 executed");
    thread t2(task2, "Task 1 executed");
    //let main wait for t1 and t2 to finish.
    t1.join();
    t2.join();
}

Rcpp

Dual core

Concurrency and parallel programming

  • Multi threaded applications through             .
#include <string>
#include <iostream>
#include <thread>

using namespace std;

//The functions we want to make the thread run.
void task1(string msg)
{
    cout << "task1 says: " << msg;
}

void task2(string msg)
{
    cout << "task1 says: " << msg;
}

//Main loop.
int main()
{
    thread t1(task1, "Task 1 executed");
    thread t2(task2, "Task 1 executed");
    t1.join();
    t2.join();
}

Rcpp

Parallel computing

  • Parallel computing is extremely simple to implement from R. 

 

# install.packages('foreach'); install.packages('doSNOW')
library(foreach)
library(doSNOW)
cl <- makeCluster(2, type = "SOCK")
registerDoSNOW(cl)

getDoParName()


#matrix operators
x <- foreach(i=1:8, .combine='rbind', .packages='wholebrain' ) %:%
   foreach(j=1:2, .combine='c', .packages='wholebrain' ) %dopar% {
     l <- runif(1, i, 100)
     i + j + l  
   }

R package

  • Why R?
    • Standard data analysis:
      • load some data
      • estimate the density distribution.
      • plot it
xx <- faithful$eruptions
fit <- density(xx)
plot(fit)

R package

  • Why R?
#Line 1: loading
xx <- faithful$eruptions
#Line 2: estimate density
fit1 <- density(xx)
#Line 2: draw 10'000 bootstraps
fit2 <- replicate(10000, {
    x <- sample(xx,replace=TRUE);
    density(x, from=min(fit1$x), to=max(fit1$x))$y
})
#Line 3: compute 95% error "bars"
fit3 <- apply(fit2, 1, quantile,c(0.025,0.975))
#Line 4: plot the estimate
plot(fit1, ylim=range(fit3))
#Line 5: add estimation error as shaded region
polygon(c(fit1$x,rev(fit1$x)), c(fit3[1,], rev(fit3[2,])), col=’grey’, border=F)
#Line 6: add the line again since the polygon overshadows it.
lines(fit1)

What other language can do this in 6 lines of code?

scRNA-seq

Gene specificity

about ~24'000 genes expressed in the brain. 

\text{Let us define the following variables.}
Let us define the following variables.\text{Let us define the following variables.}
c : \text{a unique single cell.} \quad \text{Where: } c \in \{1, ... , C\}, \text{ and } C = 380.
c:a unique single cell.Where: c{1,...,C}, and C=380.c : \text{a unique single cell.} \quad \text{Where: } c \in \{1, ... , C\}, \text{ and } C = 380.
m : \text{a unique single gene.} \quad \text{Where: } m \in \{1, ... , M\}, \text{ and } C = 380.
m:a unique single gene.Where: m{1,...,M}, and C=380.m : \text{a unique single gene.} \quad \text{Where: } m \in \{1, ... , M\}, \text{ and } C = 380.
D : \text{a } C \times D \text{ read count matrix.}
D:a C×D read count matrix.D : \text{a } C \times D \text{ read count matrix.}
D_{c,m} : \text{ normalized number of reads mapped to cell } c \text{ for gene } m.
Dc,m: normalized number of reads mapped to cell c for gene m.D_{c,m} : \text{ normalized number of reads mapped to cell } c \text{ for gene } m.
r_{c,m} : \text{ cell-gene specificity ratio.}
rc,m: cell-gene specificity ratio.r_{c,m} : \text{ cell-gene specificity ratio.}
r_{c,m} = \frac{D_{c,m}}{ s_m }
rc,m=Dc,msmr_{c,m} = \frac{D_{c,m}}{ s_m }
s_{m} = \frac{1}{C} \sum_{i=1}^C D_{i,m}
sm=1Ci=1CDi,ms_{m} = \frac{1}{C} \sum_{i=1}^C D_{i,m}
s_{m} : \text{ gene specificity.}
sm: gene specificity.s_{m} : \text{ gene specificity.}
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