Mate Pair Library Sequencing enables the generation of libraries with inserts from 2 to 5 kb in size. These long-insert Paired-End libraries are useful for a number of applications, including De Novo Sequencing, genome finishing, and structural variant detection. Combining data generated from Mate Pair library sequencing with that from short-insert paired-end reads provides a powerful combination of read lengths for maximal genomic sequencing coverage across the genome.

Following DNA fragmentation, 2-5 Kb fragments are end-repaired with biotin labeled dNTPs. The DNA fragments are circularized, and non-circularized DNA is removed by digestion. Circular DNA is fragmented and fragments biotin labels (corresponding to the ends of the original DNA ligated together) are affinity purified. Purified fragments are end-repaired and ligated to Illumina Paired-End sequencing adapters.

Additional sequences complementary to the flow cell oligonucleotides are added to the adapter sequence with tailed PCR primers. The final prepared libraries consist of short fragments made up of two DNA segments that were originally separated by several kilobases. These libraries are ready for paired-end cluster generation followed by paired-end sequencing utilizing the Genome Analyzer_IIx system.

Highlights

• High Genomic Diversity: Efficient protocol enables the highest genomic diversity of any next-generation platform
• User-Friendly Workflow: Simple two-and-a-half day workflow with limited hands-on time and multiple stopping points
• Low DNA Input Requirements: Requires as little as 10 μg of starting material