Low Density Marker Assays (LDMA) for High Throughput, Highly Multiplexed Detection, CNV, Methylation, and Expression Assays

Focused next generation sequence data can be used for the high throughput, cost effective analysis of sequences present in relative amounts (gene expression and copy number variation).  Our low density marker assay (LDMA) is a highly multiplexed ligation-dependent PCR (LD-PCR) that uses sample barcodes added by PCR to prepare a library suitable for next generation sequencing.  The sequence of the reads provides the sample and loci identification and the number of reads of each sequence provides their relative quantity in the sample.  A single sample reaction requires less than 20ng of DNA or RNA and can interrogate hundreds of sequences.  Probe hybridization, ligation and the sample indexing PCR can be carried out in a single well of a 384-well PCR plate.  Library construction containing hundreds of samples can be completed within 24 hours and data is generated on a single lane of an Illumina GAIIx flow-cell (or other instrument).  The single lane of short sequence reads generates sufficient data for the quantitative analysis of hundreds of sequences in hundreds of samples. Examples of the quantitative aspect of LDMA’s applications in: (i) gene expression, (ii) methylation status, (iii) copy number variation, (iv) presence of specific bacteria in samples and (v) others will be shown.  The basis for this wide set of applications lies in the mechanics of the assay. 

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