Figure 1

Massively parallel sequencing of samples enriched for exons on chromosome X following addition of index primers to allow multiplexing. (A) Workflow of procedure showing library construction, adaptor/index ligation, amplification and target enrichment. (B) Plot of number of reads per base across region of the chromosome X showing highly focussed sequence reads around exons and low background outside the target regions. (C) The box-and-whisker plot to examine the distribution of read coverage (log 2 of reads per base) for each of the 8 samples in uniplex, duplex and pentaplex sequencing lanes. The x-axis represents the individual lanes, while the y-axis represents the number of superimposed reads. Boxes represent the interquartile range, with the 75th percentile at the top and the 25th percentile at the bottom. The line in the middle of the box represents the 50th percentile, or median. Whiskers represent the rest of the distribution, with their terminations representing the lowest and highest feature intensity values. Box-and-whisker plots were performed for each sample in either level of plexity. (D) Effect of varying the confidence parameter in GATK analysis software on total number of SNPs called (blue line) and number of SNPs annotated in dbSNP database (red line). Representative example shown is sample D1 from the pentaplex. (E) Confirmation of DNA fragmentation and library construction by agarose gel electrophoresis confirming size range of each index amplified library.