Automated Emulsion-PCR and Sequencing of 409 Gene Panel Using Ion Proton

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SUPPLEMENTARY METHODS

Automated emulsion-PCR and sequencing of 409 gene panel using Ion Proton.

For each sample after quantitation of the library the four library pools were diluted to 200pM concentrations and combined in equal volumes to obtain the complete library pool at 200 pM concentration. This pool was further diluted to a final concentration of 12pM. Equal volumes of 12pM barcoded libraries of 6 samples were combined to obtain a multiplexed 12pM sample which was then subjected to emulsion PCR in order to amplify the library on the ISPs. The Ion OneTouch 2 System was used to break the emulsion and to isolate ISPs. Efficiency of the emulsion PCR by estimation of the ISPs with DNA was performed using the Qubit IonSphere Quality control kit (Life Technologies). Enrichment or selective isolation of ISPs with clonally amplified DNA was achieved using the Ion Torrent OneTouch ES and the IT OneTouch 200 Kit V2 (Life Technologies) as per the manufacturer’s protocol. The enriched ISPs were loaded on a Proton I chip and sequenced on Ion Proton instrument using the Ion PI sequencing 200Kit V2. A cutoff of 6 million reads with a quality score of AQ20 (1 misaligned base per 100 bases) was used as a measure of successful sequencing. Sequencing coverage of 250X and a 10% variant frequency in a background of wild type (WT) were used as minimum requirements for variant calling.

Copy number variation (CNV) detection using 409-gene panel and Ion proton.

For a population with z samples, the population average coverage depth at each amplicon is defined by:

For an individual sample, the normalized coverage ratio (ncr) is calculated for each gene with y amplicons by:

CNV analysis by Molecular Inversion Probe (MIP) Array.

80ng (6.6L at 12ng/L) of FFPE DNA, estimated by Qubit DNA HS assay (Life Technologies), was incubated with the biotin labelled molecular inversion probes overnight at 58C. Each probe is designed against a specific genomic area with a stretch of sequence complementary to that genomic region along with a distinct sequence tag non-complementary to the human DNA, which is used to hybridize to the tag probe on the array.The complementary regions span both ends of the probe and are designed to form a loop when hybridized, with a single base gap between the two ends. DNA annealed with the probes is split into two aliquots and subjected to gap-filling by using a mixture of either (dATP+dTTP) or (dGTP+dCTP). Subsequently, the DNA is subjected to exonuclease cleavage to degrade genomic DNA and probes without gap filling, thus leaving only the gap-filled circularized probe DNA intact. The circularized DNA is subjected to a single endonuclease cleavage which generates fragments with common regions on the 3′ and 5′ ends, which are used to amplify the fragments further. Post amplification, the fragments are subjected to HaeIII endonuclease digestion and hybridized to the array overnight, stained and scanned. Results from both arrays are combined by the software OncoScan Console (Affymetrix) and subjected to further analysis and visualization by Nexus Express Software V7.0 (BioDiscovery, El Segundo, CA).