04. Polony Sequencing

Polony sequencing, developed by George M. Church at Harvard Medical School, is a sequencing technique that uses paired-tag library emulsion PCR to amplify the target DNA, and sequencing by ligation to detect DNA bases. This is a combination of concepts we covered in the two previous pages.

George M. Church
George M. Church, looking magnificent.

When polony sequencing was published was released in 2003, and the cost was less than 10% of Sanger Sequencing. It was used to sequence a full E. coli genome in 2005 with an error rate of less than 0.00001%.

Open-source means freedom

One unique aspect of polony sequencing is that its technology is an open-source platform. This means the software and protocols are free and don't require licensing or a fee for use. Any modifications or improvements to the system are also made available. Additionally, the only machinery required is a computer-controlled fluidics system and an epifluorescence microscope.

Procedure

The procedure takes a total of 9 steps, but the most important parts (emulsion PCR and sequencing by ligation) were already covered in an earlier lesson.

1) Shearing DNA

The first step, as in any other NGS technique, is the library construction. We break apart the genomic DNA.

2) DNA Repair

Next we want to perform end-repair to fix any damaged or incompatible edges. We want to make our DNA ends blunt-ended with a phosphate group attached at the 5'. This allows us to ligate any adapter oligonucleotides.

The DNA fragments also undergo A-tailed treatment. This adds an A to the 3' end of the sheared DNA.

Blunt ends vs. sticky ends.
The left dsDNA has blunt ends, while the right has sticky ends.

After the DNA molecules are repaired, those of length 1kb are selected by loading them onto a 6% TBE PAGE gel.

3) DNA circularization

The next step is to circularize the DNA. We do this with the T-tailed 30 bp long synthetic oligonucleotides (T30). This contains two outward-facing Mmel recognition sites.

Restriction Enzymes

Restriction enzymes are biomolecules that are able to recognize a specific sequence and cut either at that particular spot, or a spot a certain nucleotides away from it. The cuts may be "sticky," or "blunt" depending on the type of restriction enzyme.

Circularization with T30.
Circularization with T30.

4) Rolling circle replication

The circularized DNA undergoes rolling circle replication. This is a type of nucleic acid replication that rapidly synthesizes multiple copies of circular molecules of DNA.

Rolling Cycle Amplification to generate several copies of the circularized DNA.
Rolling Cycle Amplification to generate several copies of the circularized DNA.

The newly generated circularized DNA are then digested by restriction enzyme Mmel (type IIs restriction endonucleases), which cut at a distance away from its recognition site. This releases the T30 fragment, flanked by 17-18 bp tags of the sequence (70 bp in total).

Digest the circularized DNA with Mmel, which cut a specific number of bp away from the recognition site.
Digest the circularized DNA with Mmel, which cut a specific number of bp away from the recognition site.

5) DNA Repair and primers added

The resulting DNA is repaired and FDV2 and RDV2 are added on each ends. In total, this results in a 135 bp library molecules.

We now have DNA templates with 44 bp FDV sequence, a 17-18 bp proximal tag, the T30 sequence, a 17-18 bp distal tag, and a 25 bp RDV sequence.

Attach primers FDV2 and RDV2 for emulsion PCR.
Attach primers FDV2 and RDV2 for emulsion PCR.

6) ePCR

ePCR is used to amplify the 135 bp paired end-tag library molecules. This process takes place within a water droplet embedded within an oil solution. Check out our more thorough explanation on emulsion PCR.

Product of emulsion PCR - beads with amplified DNA on surface.
This figure shows a single bead in an water-in-oil emulsion. After emulsion PCR is performed, thousands of these beads are extracted and placed inside the sequencing platform. Figure adapted from Andy Vierstraete.

7) Coverslip arraying

Coverslips are washed and treated with aminosilane. This eliminate fluorescent contamination and allows for covalent coupling of template DNA and beads to attach.

The resulting beads from ePCR are mixed with acrylamide and poured into a teflon-masked microscope slide. The coverslip is placed on top of the acrylamide gel for 45 minutes to allow for polymerization.

The beads bind to the aminosaline coating of the coverslip, spreading out in a monolayer in an acrylamide gel. The coverslip with the gel, beads and template DNA are inverted. Now beneath this solution is where the sequencing reagents will flow.

Coverslip and aminosilane.
The beads bind to the aminosilane coating to the coverslip and spread out in a monolayer in an acrylamide gel.

8) DNA sequencing

The methods for DNA sequencing is sequencing by ligation. In short, a series of anchor primers are hybridized to the synthetic oligonucleotide sequences at the genomic DNA sequences.

A group of degenerate nonamers (oligonucleotides of length 9) are used, each with a particularly known query position and fluorescent marker. Thus, in this round the known query is at position 9:

Depending on which nonamer binds, we can see which nucleotide is at position 9. We can then do this again to get the nucleotide at postition 18, then 27, and so on. Now we can use a pool of nonamers that have a known query position down one nucleotide:

We may either use these, or simply shift the known nucleotide position up one base pair and again use nonamers of known query position 1.

We perform throw in this pool of degenerate nonamers again to see nucleotides at positions 8, 16, 24, 32 and so on. We repeat this over again with different known query positions until we are through with the sequence.

Cons

  • There may be failures in cleaving the dyes, which can mess up base calls.

Pros

  • Cheap, opensource, free software.
  • Flexible. can include BAC (bacterial artificial chromosome) and bacterial genome resequencing, as well as SAGE (series analysis of gene expression) and barcode sequencing.
  • Easy to set up. Only need commonly fluorescence microscope, and a computer controlled flowcell.
  • Scalable by using 1 um magnetic beads.

References

  1. Wikipedia - Polony Sequencing
  2. Shendure J, Porreca GJ, Reppas NB, Lin X, McCutcheon JP, Rosenbaum AM, Wang MD, Zhang K, Mitra RD, Church GM (2005). "Accurate multiplex polony sequencing of an evolved bacterial genome". Science 309 (5741): 1728–32.

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