Multiplex Polymerase Chain Reaction

Activated memory B cell supernatants were screened in a high throughput format for neutralization activity using a micro-neutralization assay, as described^{2 (link)}. Heavy and light chain variable regions were isolated from B cell lysates of selected neutralizing hits by reverse transcription from RNA followed by multiplex PCR amplification using family-specific V-gene primer sets. For some antibodies, traditional cloning methods were used for antibody isolation, as described^{2 (link)}. For other antibodies, amplicons from each lysate were uniquely tagged with multiplex identifier (MID) sequences and 454 sequencing regions (Roche). Single round of replication pseudovirus neutralization assays and cell surface binding assays were performed as described previously^{2 (link),27 (link),28 (link)}. Glycan reactivities were profiled on a printed glycan microarray (version 5.0 from the Consortium for Functional Glycomics (CFG)) as described previously^{29 (link)}.

We evaluated the COSMIC (Bamford et al, 2004 (link)) database and PubMed to select a panel of genes and loci previously reported to be frequently affected by somatic mutation in human cancer. We chose 13 cancer genes and designed 58 assays to test for individual mutational events, which included: 1 insertion, 3 deletions and 52 substitutions (Supporting Information Table S1). Genomic position and sequencing information for all mutation sites were collected using the RefSeq gene sequences obtained using the human genome browser from the University of California Santa Cruz (UCSC), NCBI build 36.1. Primers for multiplexed PCR amplification were designed using Primer 3 software. Since FFPE tissue can be highly fragmented and of poor quality, design parameters restricted amplicon length to a maximum of 200 nt. All amplification primers (Supporting Information Table S5A) include a 10 nt long 5′ anchor tail (5′-ACGTTGGATG-3′) and the final PCR products range in length between 75 and 187 nt. The extension primer probes (Supporting Information Table S5B) were designed manually, according to the ABI PRISM SNaPshot Multiplex Kit protocol recommendations (Life Technologies/Applied Biosystems, Foster City, CA) and using primer analysis tools available through the Primer 3 and Integrated DNA Technologies (IDT, Coralville, IA) web interfaces. Optimal conditions for multiplexed assays were determined empirically and are summarized in Supporting Information Table S6.
As part of the design rationale, we included assays covering four adjacent loci that are commonly mutated in the therapeutically relevant KRAS and NRAS oncogenes (for both genes we are targeting nucleotide positions: 34G, 35G, 37G and 38G). Due to the close proximity of these sites, to avoid compromising assay sensitivity due to primer competition, we decided to assay each of them in an independent panel. In addition, due to the extreme sequence similarity between KRAS and NRAS, to avoid non-specific results, we segregated the assays for the two genes into individual multiplexed reactions. We thus started with eight panels, which were populated with the 58 assays outlined in Supporting Information Table S1. Many of these genes and assays are clinically relevant. In addition, since the costs of running the assay (regarding tumour material and the actual price per assay) are mainly dictated by the number of panels, we decided to also include a set of common mutations affecting critical cancer genes for which a therapeutic agent is still currently unavailable. We hope that the addition of these less ‘clinically relevant’ mutations will still be useful in a clinical setting, as we may find them to correlate with a better or worse prognosis or to influence response to specific therapies, and thus contribute to better cancer care in the future.