A Balanced Barcoding System for Multiplexed DNA library and SOLiD SAGE Sequencing

摘要

>RP-27A set of 96 molecular barcode adaptors specifically designed for the SOLiD™ platform have been validated for use with DNA fragment and paired end libraries. Moreover, the barcode system is adapted for multiplexed Serial Analysis of Gene Expression (SAGE). DNA libraries are constructed with a multiplex adaptor which consists of three segments: (1) an internal sequencing primer binding site, (2) a barcode decamer sequence and (3) a P2 PCR priming site. The barcode and target DNA are then sequenced as two separate reads from the same strand allowing for the libraries to be pooled in a multiplexed emulsion PCR and deposited into a single spot on a SOLiD™ slide. Similarly, SAGE libraries are constructed with a modified adaptor allowing for the addition of unique barcode primers with a short cycle amplification consistent with the SOLiD™ barcoding system. The modular barcoding design requires only 5bp of sequencing to distinguish 16-plex samples and 10bp of sequencing to distinguish 96-plex samples. The barcodes are optimized in sets of four wherein each set is color balanced at every position. Importantly, clear discrimination between barcode samples is achieved by maintaining a minimum Hamming distance of 3 colorspace calls for optimal data integrity. The DNA barcode system was validated by sequencing of E. coli fragment libraries. Error rates and quality value (QV) scores for the barcode reads were found to be consistent across the final set. Importantly, QV scores were also consistent for the reads, indicating minimal effects of the barcode decamers on bead templating and ligation sequencing efficiency. Furthermore, the set of 16 SAGE barcoded samples yielded Pearson correlations above 0.98. Ongoing development studies include integration with methods of target enrichment that will further enable high levels of DNA and RNA expression library multiplexing afforded by the increasing throughput of the SOLiD™ system.