41J Blog

Sequencing system image from patent.

Building on my list of sequencing companies. I’ve put together a few brief notes on Genapsys.

Business

Genapsys was founded in 2010. They have raised in excess of 84M USD in total (110M USD according to their website, 84M accounted for on crunchbase). Investors appear to include Decheng Capital, IPV Capital, Plug and Play Ventures and possibly Ampersand Capital Partners. Yuri Borisovich Milner (DST Global) is also said to be an investor. Their last round was on January 2018, where they raised 32.5M USD, Series C. In addition to venture funding they have received some grant funding (~4M USD) [1].

The company was founded by Dr. Hesaam Esfandyarpour and incubated at the Stanford Genome Technology Center.

Technology

The patents I’ve reviewed describe two key components to the Genapsys approach. The first is a method for confining DNA and reagents using Virtual Wells [3]. The second is a method for detecting base incorporation (to build a single channel sequencing-by-synthesis platform).

Virtual Wells

One of the key elements of the 454 and Ion torrent platforms was their use of wells to confine beads on an array (and in Ion torrents case over a sensor). Both these platforms used an off chip process to amplify DNA on the bead (emulsion PCR). This added an extra and somewhat awkward step to the sequencing process.

The Genapsys approach suggests doing away with wells completely. Instead magnetic and electric fields would be used to confine beads and reagents. Magnetic beads would be used which would be attracted to magnets on the chip, localising the bead over sensors.

They then suggest using electric fields to confine nucleotides, strands, enzymes and potentially other reagents. Using the virtual well technology the amplification process could take place on chip. I assume this would be the same process as used for emulsion PCR. However, rather than the reaction vessel being a tiny water bubble, the charged reagents are confined by the electric field.

This seems like a rather neat idea, but I’m curious to know how well it works in practice. I would guess confinement is not perfect.

Detection

Initial reports all suggested that incorporation would be detected either through changes in pH (ISFET) or heat [2]. While recent patents still mention pH, heat, and charge based detection, they also discuss detection of incorporation through conductivity and impedance changes in the Debye layer of the bead [5].

The Debye layer as far as I can tell just means the double layer. My understanding is that in this scenario the bead will be negatively charged. There will therefore be a double layer formed around the bead:

By placing electrodes on either side of the bead you can measure conductivity and impedance/capacitance. I would imagine that most current will come from the bulk of the solution, however the patents suggest that contribution from the double layer can be measured.

Exactly how additional nucleotides effect the conductivity/impedance of the double layer is less clear to me. As they will increase the negative charge in the vicinity of the bead it seems logical that they would however.

If it really is just the additional charge they add, the system feels similar to other methods of detecting the beads overall charge (like the caerus approach). However perhaps by looking at the change in double layer current background contributions are reduced, or the effect of the charge difference is amplified.

One patent does show “sequencing data”, as far as I can tell this is more likely to have come from simulation than a real experiment, I’ve highlighted a large deflection caused by multiple incorporation in the figure below:

Another factor that leads me to believe they may be pursuing a bead based, Debye layer based approach (rather than using ISFETs) is that pairs of electrodes are shown along side beads repeatedly.

There are a few images of seemly real chip systems, but I didn’t see any SEM images. One example is this figure showing bead occupancy (which looks pretty good!):

Overall, while Genapsys haven’t released a whole lot of public information, the patents seem to give a reasonable indication of what they’re working on. Charge based approaches seem attractive. One advantage they have over other chip based approaches is that you don’t need to monitor the incorporation in real time. Bases can be incorporated, and the charge difference measured at low speed, and potentially under different reagent conditions.

However, while patents suggest chips maybe reusable, you only get a single read from each “virtual well”. As with Ion torrent and 454, this could ultimately limit throughput.

Overall, some interesting ideas. And I look forward to seeing how things pan out.

Notes

[1] http://grantome.com/grant/NIH/R01-HG006889-03

[2] Genomeweb article: https://www.genomeweb.com/sequencing/genapsys-develop-microelectronic-sequencer

“DNA sequencing method that is based on direct heat or pH measurement”

US Patent No. 7,932,034, “Heat and pH measurement for sequencing of DNA.”

“In 2010, the firm won a $250,000 grant through the Qualifying Therapeutic Discovery Project Program for a project entitled “Development of an inexpensive, ultra-high throughput micro-electronic medical sequencer””

[3]

http://www.freepatentsonline.com/9399217.html
“As used herein, “virtual wells” refers to local electric field or local magnetic field confinement zones where the species or set of species of interest, typically DNA or beads, generally does not migrate into neighboring “virtual wells” during a period of time necessary for a desired reaction or interaction.”

Virtual Well

The a virtual well or “chamber-free array”, may detect or manipulate particles (e.g., beads, cells, DNA, RNA, proteins, ligands, biomolecules, other particulate moieties, or combinations thereof) in an array wherein said array captures, holds, confines, isolates or moves the particles through an electrical, magnetic or electromagnetic force and may be used for a reaction and or detection of the particles and or a reaction involving said particles. Said “virtual well” may provide a powerful tool for capturing/holding/manipulating of beads, cells, other biomolecules, or their carriers and may subsequently concentrate, confine, or isolate moieties in different pixels or regions of the array from other pixels or regions in said array utilizing electrical, magnetic, or electromagnetic force(s). In one embodiment the array is in a fluidic environment. Sensing may be done by measurement of charge, pH, current, voltage, heat, optical or other methods.

[4] 2013 patent: http://www.freepatentsonline.com/20130096013.pdf
ISFET, chemFET or nanobridge.

[5] Recent patent: http://www.freepatentsonline.com/y2018/0119215.html
“detect a change in conductivity within a Debye layer” ” can detect nucleotide incorporation events by measuring local impedance changes of the magnetic beads 220 and/or the amplified DNA (or other nucleic acid) 255 associated with the magnetic beads 220. Such measurement can be made, for example, by directly measuring local impedance change or measuring a signal that is indicative of local impedance change. In some cases, detection of impedance occurs within the Debye length (e.g., Debye layer) of the magnetic beads 220 and/or the amplified DNA 245 associated with the magnetic beads 220. Nucleotide incorporation events may also be measured by directly measuring a local charge change or local conductivity change or a signal that is indicative of one or more of these as described elsewhere herein. Detection of charge change or conductivity change can occur within the Debye length (e.g., Debye layer) of the magnetic beads 220 and/or amplified DNA 245 associated with the magnetic beads 220.”

“using the sensor to detect a change in conductivity within a Debye layer of the bead upon incorporation of at least one nucleotide of the population of nucleotides into a growing nucleic acid strand, which growing nucleic acid strand is derived from the primer and is complementary to the nucleic acid template”

A previous post discussed Cygnus’ approach to sequencing, using mixtures of bases and multiple reads of the same template. Centrillion also have a patent that appears to cover a related approach.

The Cygnus approach, as described in their paper uses mixtures of 2 bases. I thought it might be interesting to work through corrections using mixtures of 3 bases. It’s possible this is covered somewhere in their supplementary info, or huge 200+ page patent. I’ve not checked and this is just for fun.

There are 4 possible sets of 3 different base types: ATG, ATC, TGC and AGC. The difference between each of these sets is clearly a single base (3 bases out of ATGC in the set, and 1 left out).

To recap on the previous post, a template is exposed to alternating sets (mixtures) of bases, and we measure incorporation intensity and learn how many bases incorporate (as in the same for a normal single channel unterminated sequencing chemistry). In order to process the entire strand the sets we alternate between must contain all base types. For the sets of 3 base types this is no problem, any pair of sets will contain all four base types and differ by only a single base type.

There are 6 possible pairings:

a ATG,ATC

b ATG,TGC

c ATG,AGC

d ATC,TGC

e ATC,AGC

f TGC,AGC

We could vary the order of the pairs. But we don’t really need to. Working through all possible 2bp repeats [1] it’s clear that we can accurate resolve all sequences using 3 out of the 6 alternating pairs.

In all cases, one pairing supplies the base transition information. For example for the repeat ATATAT this is group f above. This is the only pairing that blocks incorporation between A and T transitions. Each pairing blocks on transitions between one of the six possible transition types (G<->C A<->T A<->G A<->C T<->G T<->C). To accurately resolve all sequences, all pairings are therefore required. In the example 2bp repeats, one pairing provides the “transition” information and 2 other pairings are required to resolve the sequence to one of the four bases.

You therefore need to sequence each template six times. However, at any given base information from only 3 of the “mixture sequences” is required to resolve the strand. The other 3 sequences provide redundant information for error correction. This information could be used in a number of ways (either masking likely errored bases, taking a majority vote, or using this information in a more complex error correction model).

How much sequencing does this require as compared to standard single base sequencing?

Well, there will always be degenerate sequences, both in this scheme and the Cygnus approach. These sequences will require very slightly more sequencing than using a normal single base incorporation system.

However we can simulate the number of cycles required (a cycle being the incorporation of a single base type, or a single mixture type). I quickly threw some code together to do this [2]. Assuming this hastily thrown together code is correct the single base incorporation scheme requires 1.481 cycles per base (or ~2.7 bases incorporated per set of 4 bases). The mix of 3 scheme described above requires 1.4905 cycles per base.

So, if you just go by this, there’s very little overhead.

One downside of the base mixture incorporations is that the sequencing system has to cope with longer homopolymers (or rather runs of 1 of 3 different base types). Again this is true of the approach described here, and the Cygnus system. What issues this causes, will depend on the error profile of the underlying technology.

While I’ve discussed mixtures of 3 bases here, it might also be interesting to look at combinations of mixtures of 2 and 3 bases. For example you might have set pairs of ATG, and ATC. Then a set of CA and GT to resolve the ambiguity (this could be extended to create a complete sequencing system).

Maybe that’s another fun project for another time.

Notes

[1]

[2]

#include <iostream>
#include <vector>
#include <math.h>
#include <stdlib.h>

using namespace std;

// Multiple base incorporations
string s1 = "ATG";
string s2 = "ATC";
string s3 = "TGC";
string s4 = "AGC";

int mix_incorp(string temp,vector<string> pair) {

  int p=0;
  int cycles=0;
  for(int n=0;n<temp.size();) {
  
    for(;;) {
      bool ad=false;
      if(temp[n] == pair[p][0]) {n++; ad=true;}
      if(temp[n] == pair[p][1]) {n++; ad=true;}
      if(temp[n] == pair[p][2]) {n++; ad=true;}
      if(ad==false) break;
    }

    cycles++; 
    if(p==0) p=1; else p=0;
  }

  return cycles;

}

int main() {

  string temp;

  // generate random sequence
  for(int n=0;n<10000;n++) {
    int r = rand()%4;
    if(r == 0) temp += "A";
    if(r == 1) temp += "T";
    if(r == 2) temp += "G";
    if(r == 3) temp += "C";
  }

  cout << "Sequence: " << temp << endl;

  // Single base incorps
  string order="ATGC";
  int pos=0;
  int cycle_count=0;
  for(int n=0;n<temp.size();) {

    for(;temp[n] == order[pos];) n++;
   
    pos++;
    cycle_count++;
    if(pos == order.size()) pos=0;
  }
  cout << "Average cycles per base, single base incorps: " << ((float)cycle_count)/((float)temp.size()) << endl;

 
  // Super ugly code, but functional...
  vector<vector<string> > pairs(6);
  pairs[0].push_back(s1); 
  pairs[0].push_back(s2); 
  pairs[1].push_back(s1); 
  pairs[1].push_back(s3); 
  pairs[2].push_back(s1); 
  pairs[2].push_back(s4); 
  pairs[3].push_back(s2); 
  pairs[3].push_back(s3); 
  pairs[4].push_back(s2); 
  pairs[4].push_back(s4); 
  pairs[5].push_back(s3); 
  pairs[5].push_back(s4); 
  
  int total=0;
  for(int n=0;n<6;n++) {

    int count = mix_incorp(temp,pairs[n]);
    total+=count;
  }
  cout << "Average cycles per base, mixture incorps: " << ((float)total)/((float)temp.size()) << endl;
}

Yesterday I wrote up my notes on Iridia. I think one of the things I find so fascinating about the concept is the potential to create a system that can both read and write DNA. This move to read/write DNA devices seems like a step change from discrete sequencers and synthesizers.

To briefly recap, the Iridia system used nanopore to selectively expose DNA strands to enzymes. The enzymes can’t make their way through the pore, but you can drive the charged DNA strand through the pore under a bias voltage.

The use of a nanopore also clearly lends itself to using this same aperture for sequencing the DNA. This could be through the detection of the blockage of an Ionic current, or embedded electrodes (like some solid state nanopore systems). Either solid-state, or protein nanopores could be used. Apertures might even be constructed in other ways (like Armonica’s tortuous nanopores).

So potentially, you have a DNA synthesis platform, that can also QC the strand at every base incorporation as it passes through from one chamber to another.

One issue with at least some embodiments of the Iridia concept from their patent, is that single nucleotides can make their way through the pore as well was the strand under synthesis. This means they need to remove the terminator from the base already incorporated into the strand only (I think they may have a way of doing this). I guess it can also increase the potential for misincorporation and might complicate the fluidic system.

However it feels like if the correct components can be assembled, you might be able to create a chip system that can read and write DNA and has no external fluidic components.

I thought it might be fun to play around with these ideas a bit…

The diagram below shows what this (Computer Scientist) imagines such a system might look like schematically:

In the above diagram I’ve shown a system with multiple chambers. Each chamber has two apertures shown. One is a nanopore/nanoscale aperture through which only DNA can pass. The other is a valve, which stops all flow into the chamber. The valve can be much bigger than the nanopore, these are all normally closed. Potentially the valve could be part of the pore (for example a voltage gated ion channel [2]) or might not be required at all. But this valve could be large and should be easy to fabricate.

The strand under synthesis sits in the input DNA chamber. You then open the value leading out of this chamber (1) and going to one of the nucleotides, (2) for example. A bias voltage is applied between these chambers to drive the DNA into the correct nucleotide chamber. In this chamber there’s a template independent polymerase. The nucleotides in this chamber are terminated, such that only a single nucleotide is incorporated into the strand.

The bias voltage is then reversed, and the strand flows back into the “input” chamber. A number of single nucleotides come along for the ride. However in the input chamber the strand is captured. This could be by hybridising to an immobilized complementary strand, though there might be other methods. With the strand captured you reverse the bias voltage again and the single nucleotides flow back into their original chamber. You can then close the valves to keep them contained. The strand is released by heating the strand, melting it.

Next you need to cleave the terminator. This will depend on the type of terminator used. One possibility might be to use photo-cleavable terminators such as those developed by LaserGen [1]. If this is the case, then you would just need to turn on a light source to remove the terminator. Another chamber could be used however, particularly if there is some other process (perhaps there’s an enzymatic process?) that is used to remove the terminator.

The process would then continue as above cyclically. Depending on the quality of the pores/apertures, you can also measure the ionic current as the strand passes through the pore. This may be sufficient to determine the sequence during synthesis.

In any case, once synthesis is complete you can open another valve (7) and set bias voltages between the chambers (input and holding for QC chambers) such that the DNA will flow through an exit pore. This pore could be specifically designed for sequencing and might have additional embedded electrodes to enable this.

You could also QC the strand (did synthesis complete correctly?) and sort the strand accordingly.

Of course, the same system could also be used as a read system only, just insert complete strands at the start of the process.

Potentially all the nucleotides and reagents could come preloaded, giving a simple, almost solidstate system. While initially it might be desirable to fabricate only certain parts of the system using nanofabrication, ultimately it might be possible to integrate all components onto a single chip.

Notes

[1] https://webcache.googleusercontent.com/search?q=cache:hkUhGFAhmmgJ:https://www.genomeweb.com/sequencing/lasergen-says-its-new-reversible-terminators-could-improve-several-sequencing-pl+&cd=15&hl=en&ct=clnk&gl=jp

[2] https://en.wikipedia.org/wiki/Voltage-gated_ion_channel

While putting together my list of synthesis companies, one particular stood out. Not least because of its original name, Dodo Omnidata (which is awesome) [3]. But also because the technology is significantly different from anything else on the list being inherently single molecule. The company also seems to be relatively unknown.

For these reasons, I’m writing up some quick notes.

Business

Dodo Omnidata was founded in 2016. They seem to have raised ~400K in seed funding in 2017. An SEC filing shows they raised ~2MUSD this June. Jay Flatley (ex-Illumina CEO) is on the board. The initial 400K came from Tech Coast Angels according to Crunchbase. It’s not clear where the most recent raise came from, but with Jay on the board, it seems possible there’s a connection to Illumina Ventures.

Technology

There’s not much on the website, but there is a 134 page patent. I’ve barely skimmed it but what’s clear is that they suggest using nanopores for DNA synthesis:

From by quick skim, it appears that what they suggest is driving a strand of DNA through a nanopore with a bias voltage. In this way they can move it between two chambers. In itself I don’t believe that is particularly novel. What’s neat is that because enzymes are too big to go through the nanopore they can selectively expose the strand to different enzymes under electrical control.

They use this for synthesis by having one chamber containing a template independent polymerase (a polymerase that just adds any base you give it) and a base with a terminator on it (so only a single base is added). My guess is that you’d flow bases in cyclically. If you want to incorporate a base into the strand, you flip the voltage and pull the strand through the nanopore. Leave it for a while to incorporate the base, then pull it back out.

Back on the other side of the pore, another enzyme comes in and removes the block on the strand. As single nucleotides can also pass through the pore, it’s desirable to have an enzyme that only removes terminators on bases incorporated into the strand.

In practice I would imagine the whole system can be arrayed. And you’d be flowing bases onto one side of an array. How competitive this system is with other enzymatic approaches is something I don’t know. But it seems pretty neat!

Notes

[1] 2018 SEC Filing: https://www.sec.gov/Archives/edgar/data/1708118/000170811818000002/0001708118-18-000002-index.htm

[2] http://www.freepatentsonline.com/WO2017151680A2.pdf

[3] In case you’re curious about the binary encircling the old Dodo Omnidata logo it converts to Data Vida in ASCII. Vida is Spanish for life, and I assume is a reference to the tagline “Data for Life” also on their banner.