Abstract 2186: Error correcting DNA barcode allows for high throughput mutation screening in patient cohorts by multiplexed high throughput sequencing
Authors: Bilke Sven , Walker Robert L. , Pineda Marbin , Killian Keith , Abaan Ogan , Meltzer Paul S. 
Published: February 2011

Somatic mutations are one of the major factors contributing to oncogenesis and tumor progression. Second generation sequencing allows rapid generation of several giga-bases of sequence, enabling genome-wide mutation discovery in individual samples. This is particularly effective when used with genome partitioning strategies. The biological significance of mutations may be assessed by their prevalence at the gene or pathway level and by their functional implications, requiring larger sample cohorts. Adding sequence barcodes to discriminate individual identity allows pooling of sample sets larger than those described by current Illumina protocols which are relatively complex and currently offer only up to 12 barcodes.

We report a novel barcoding protocol for Illumina GA2-x sequencers, which permits multiplexing up to 2048 samples per flow-cell. Reducing hands-on time in the protocol was one major goal in designing the protocol. Throughput has been increased by up to ten-fold compared to Illumina protocols. This improvement was achieved by eliminating several enzymatic steps and gel purification/size selection and replacing those with two consecutive PCR steps. Instead of enzymatically ligating of the bar-code, our protocol incorporates barcode, sequencing and flow cell binding sites into the DNA product through a universal set of oligo primers, independent of target selection. Additionally, the PCR based protocol utilized the Fluidigm Biomark system for parallel processing allowing the processing of 317 amplimers x 48 samples in parallel.

“Cross-talk” between samples and loss of usable sequences induced by read-errors in the bar-code is a major concern in multiplexed experiments. In order to minimize such effects, we introduced an error correcting 8nt DNA code using concepts from computer science. The double 8,4 Hamming code utilized guarantees error correction of read errors affecting up to 2nt, thereby boosting the number of usable codes. Additionally, error detection of read errors affecting up to 4nt minimize cross-talk between multiplexed samples.

The protocol has been validated in a 48-way multiplexed experiment demonstrating the viability of the more automated, less laborious library preparation. Error detection and correction was highly effective with the number of reads mapped to a valid bar-codes could be increased by15-20% on average. In one particularly difficult barcode, almost 60% of reads were rescued by error correction.

In summary, the protocol presented here extends the use of Illumina GA2 sequencers for mutation screening in cohorts of up to 2048 samples per experiment for a small set of target genes.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2186.