Los efectos de la difusión

The Ion-Torrent sample preparation workflow involves more complex protocols in comparison to the Illumina workflow. Nevertheless, library construction is similar wherein 1 ng to 10 μg of starting genomic DNA material is subjected to numerous processing steps: (1) DNA fragmentation, (2) end-repair and 5’-phosphorylation, (3) adaptor-ligation and nick translation and (4) emulsion-PCR enrichment. For our study, a total of five different types of sample preparation kits were used to generate an Ion-Torrent library in preparation for sequencing: 1) Ion-Xpress Plus Fragment Kit, 2) Ion-Xpress Barcode Adapters, 3) Ion- Library Quantification kit, 4) Ion-PGM template generation (OT2) 400 bp kit and lastly 5) Ion-PGM sequencing 400 bp kit.

The Ion-Xpress fragmentation kit utilises enzymatic shearing for a greater and faster shearing process. The enzyme fragmentase used in this kit shears large genomic DNA > 10 kb into 100 - 800 bp fragments depending on the incubation time; normally about six to seven minutes which is similar to that for mechanical shearing. The enzyme fragmentase has two functions, first it randomly nicks dsDNA and secondly, it binds to the nicked site and cuts the opposite DNA strand thus producing a dsDNA overhang breakage known as a “sticky-end” which needs to be repaired as soon as possible to prevent re-annealing of both the complementary strands (Liu, Z., 2010). According to the manufacturer: Life-Technologies, enzymatic shearing and mechanical shearing produce similar results in terms of size distribution and sequence coverage. An advantage of using enzymatic shearing is that the fragment size can be controlled by diluting fragmentase and by using different incubation times tailored to generate the desired sized fragments for both AT- and GC-rich genomic libraries (Liu, Z., 2010). Following fragmentation, end-repair and phosphorylation, the enzymes Klenow-exo, and T4 DNA polymerase are used to repair the sticky ends of the dsDNA, prior to DNA ligation and emulsion-PCR enrichment.

Targeted-sequence enrichment serves several important purposes, (1) it increases the amount of prepared DNA template, (2) facilitates selection of molecules that are successfully adapter- ligated, (3) enables addition of indices for multiplexing technique and (4) enables incorporation of oligonucleotide sequences for the attachment of the library to the beads. There are two steps in the Ion-Torrent workflow where PCR enrichment occurs; the first is the amplification of the adapter-ligated DNA fragments and the second is the emulsion PCR.

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45 Prior to sequencing the emulsified micro-reactors are broken apart to release the enriched DNA containing beads before being immobilized into the Ion-Chip for sequencing.

The Ion-Torrent Personal Genome Machine (PGM) was first introduced commercially in 2011. It sequences DNA by detecting the identity of the incorporated bases without the need for chemical luminescence dyes, with no requirement for camera optics, no light and no moving parts, hence making it simpler, faster and more affordable than other NGS platforms (Quail et al., 2012; Rothberg et al., 2011). Ion-Torrent uses semi-conductor chip technology that contains millions of tiny micro-wells under a huge sensing pixelated layer similar to the CMOS (complementary metal oxide semiconductor) light sensor chip found in the modern digital camera (Figure 6) (Quail et al., 2012). These tiny wells capture ionic pH changes during DNA sequencing which are then later translated to digital information. CMOS sensors are less sensitive than CCD sensors but are 10 ~ 100 times faster in processing light sources due to the ability to read and translate each pixel individually and simultaneously, producing excellent quality images with low background noises (Figure 7A). However in the Ion- Torrent PGM, the CMOS sensor has been modified and paired with an ISFET (Ion Sensitive Field Effect Transistor) sensor to sense chemical changes instead of changes in light (Figure 7B) (Rothberg et al., 2011). The sensor is positioned at the bottom layer over the electronics for transferring electrons during the transduction of voltage from the incorporation event (Figure 7B) (Rothberg et al., 2011) and is used as an independent pH monitor directly

measuring the release of a hydrogen ion (H+) during the incorporation of a nucleotide

(Rothberg et al., 2011).

The semiconductor Ion-chip is a wafer-like square made from polycarbonate that contains millions of micro wells designed to hold and control fluidics on top of a CMOS and ISFET sensor arrays for the detection of electrical signals (Figure 7C) (Rothberg et al., 2011). These miniscule wells are designed to retain fluidics within the high conductivity material to ensure efficient electrical signal transduction (Figure 7D). Presently in the Ion-Torrent PGM sequencing system there are three types of Ion-chips (Ion-314, -316 and -318 v2) and each has a different sequencing output from 30 megabases to 2 gigabases with a total number of reads ranging from 400 to 5.5 million sequences (Shokralla et al., 2012). The exponential increase in the sequencing output was achieved by increasing the diameter of the semiconductor die cast area from an original size of 10.6 mm x 10.9 mm to 17.5 mm x 17.5 mm thus increasing the density output (Figure 7E and 7F) (Rothberg et al., 2011). However

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46 the expansion of the area is limited by the CMOS sensor size and number of transistors. Further expansion would require a redesign of the Ion-chip system.

The sequencing process begins with the denaturation of the prepared DNA libraries inside the micro wells flooded with a dNTP solution. Within these wells, DNA nucleotides are incorporated one at a time via DNA polymerase. During this step whenever a nucleotide is incorporated into a single strand of the DNA, it releases a free hydrogen ion (H+) as a by- product. The alteration in pH is then translated to a voltage signal before being recorded by the pH meter inside the microchip and translated later into digital information for each incorporated nucleotide base (Rothberg et al., 2011). Since each DNA nucleotide emits a different pH reading and voltage, the nucleotides can be confidently base-called individually without much error. In the event of identical nucleotide bases next to each other the voltage signal will give a double or more signal readout when base-calling, e.g. if three bases of thymine (T) are detected then the voltage signals for thymine will be increased to three-fold on a pH voltage meter. This sequencing process occurs across millions of wells in a microchip simultaneously which explains why the sequencing process only takes few hours instead of days for chemiluminescence detection. This semiconductor approach gives a read length range from 100 to 400 bp DNA fragments (Liu, L. et al., 2012; Quail et al., 2012; Rothberg et al., 2011).

For this project we have chosen both the Illumina MiSeq NGS platforms and Life- Technologies Ion-Torrent PGM as our preferred sequencing instruments. Both chain- termination based platforms and semi-conductor chip technology are equally capable of producing massive parallel sequencing sharing similarities in workflow, engineering configurations and sequencing chemistry. In the present work we compare the results from semiconductor sequencing with those obtained from Illumina SBS system for their performance while evaluating the data quality and the associative running cost.

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Ion-Torrent PGM sequencing system

Figure 6– Ion-Torrent PGM sequencer, A) touch screen control, B) Ion-chip loading deck clamping mechanism,

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Ion-Torrent PGM sensor, well and chip design

Figure 7 – Technology behind semiconductor sequencing, A) CMOS sensor build on a wafer shape

polycarbonate die, B) underlying electronics and sensors board, C) upper surface of the Ion-chip showing location for addition of sequencing reagents, D) A schematic diagram showing the technology behind semiconductor sequencing with DNA template releasing H+ _{ions which change the pH of the well - this signal is}

transformed into potential voltage and sensed by the under lying sensor and electronics, E) electron micrograph showing connection between miniscule well and ISFET sensor, F) schematic diagram for the sensor detection workflow in two-dimensional array.

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