DNA nanoball sequencing

DNA nanoball sequencing is a high throughput sequencing technology that is used to determine the entire genomic sequence of an organism. The method uses rolling circle replication to amplify small fragments of genomic DNA into DNA nanoballs. Fluorescent nucleotides bind to complementary nucleotides and are then polymerized to anchor sequences bound to known sequences on the DNA template. The base order is determined via the fluorescence of the bound nucleotides This DNA sequencing method allows large numbers of DNA nanoballs to be sequenced per run at lower reagent costs compared to other next generation sequencing platforms. However, a limitation of this method is that it generates only short sequences of DNA, which presents challenges to mapping its reads to a reference genome. After purchasing Complete Genomics, the Beijing Genomics Institute (BGI) refined DNA nanoball sequencing to sequence nucleotide samples on their own platform. DNA nanoball sequencing is a high throughput sequencing technology that is used to determine the entire genomic sequence of an organism. The method uses rolling circle replication to amplify small fragments of genomic DNA into DNA nanoballs. Fluorescent nucleotides bind to complementary nucleotides and are then polymerized to anchor sequences bound to known sequences on the DNA template. The base order is determined via the fluorescence of the bound nucleotides This DNA sequencing method allows large numbers of DNA nanoballs to be sequenced per run at lower reagent costs compared to other next generation sequencing platforms. However, a limitation of this method is that it generates only short sequences of DNA, which presents challenges to mapping its reads to a reference genome. After purchasing Complete Genomics, the Beijing Genomics Institute (BGI) refined DNA nanoball sequencing to sequence nucleotide samples on their own platform. DNA Nanoball Sequencing involves isolating DNA that is to be sequenced, shearing it into small 100 – 350 base pair (bp) fragments, ligating adapter sequences to the fragments, and circularizing the fragments. The circular fragments are copied by rolling circle replication resulting in many single-stranded copies of each fragment. The DNA copies concatenate head to tail in a long strand, and are compacted into a DNA nanoball. The nanoballs are then adsorbed onto a sequencing flow cell. The color of the fluorescence at each interrogated position is recorded through a high-resolution camera. Bioinformatics are used to analyze the fluorescence data and make a base call, and for mapping or quantifying the 50bp, 100bp, or 150bp single- or paired-end reads. Cells are lysed and DNA is extracted from the cell lysate. The high-molecular-weight DNA, often several megabase pairs long, is fragment by physical or enzymatic methods to break the DNA double-strands at random intervals. Bioinformatic mapping of the sequencing reads is most efficient when the sample DNA contains a narrow length range. For small RNA sequencing, selection of the ideal fragment lengths for sequencing is performed by gel electrophoresis; for sequencing of larger fragments, DNA fragments are separated by bead-based size selection. Adapter DNA sequences must be attached to the unknown DNA fragment so that DNA segments with known sequences flank the unknown DNA. In the first round of adapter ligation, right (Ad153_right) and left (Ad153_left) adapters are attached to the right and left flanks of the fragmented DNA, and the DNA is amplified by PCR. A split oligo then hybridizes to the ends of the fragments which are ligated to form a circle. An exonuclease is added to remove all remaining single-stranded and double-stranded DNA products. The result is a completed circular DNA template. Once a single-stranded circular DNA template is created, containing sample DNA that is ligated to two unique adapter sequences has been generated, the full sequence is amplified into a long string of DNA. This is accomplished by rolling circle replication with the Phi 29 DNA polymerase which binds and replicates the DNA template. The newly synthesized strand is released from the circular template, resulting in a long single-stranded DNA comprising several head-to-tail copies of the circular template. The resulting nanoparticle self-assembles into a tight ball of DNA approximately 300 nanometers (nm) across. Nanoballs remain separated from each other because they are negatively charged naturally repel each other, reducing any tangling between different single stranded DNA lengths. To obtain DNA sequence, the DNA nanoballs are attached to a patterned array flow cell. The flow cell is a silicon wafer coated with silicon dioxide, titanium, hexamethyldisilazane (HMDS), and a photoresist material. The DNA nanoballs are added to the flow cell and selectively bind to the positively-charged aminosilane in a highly ordered pattern, allowing a very high density of DNA nanoballs to be sequenced. After each DNA nucleotide incorporation step, the flow cell is imaged to determine which nucleotide base bound to the DNA nanoball. The fluorophore is excited with a laser that excites specific wavelengths of light. The emission of fluorescence from each DNA nanoball is captured on a high resolution CCD camera. The image is then processed to remove background noise and assess the intensity of each point. The color of each DNA nanoball corresponds to a base at the interrogative position and a computer records the base position information. The data generated from the DNA nanoballs is formatted as standard FASTQ formatted files with contiguous bases (no gaps). These files can be used in any data analysis pipeline that is configured to read single-end or paired-end FASTQ files.