Long-Read Sequencing Technology

Moleculo, acquired by Illumina in late 2012, has developed an innovative technology for generating long reads that combines a new sample prep method and genome analysis tools. The technology breaks DNA into large fragments that are sequenced on standard Illumina sequencing systems for subsequent assembly into synthetic long reads using proprietary informatics.

The Moleculo sample prep is a full end-to-end workflow from DNA to sequencing-ready library. Applications for this long read technology include de novo assembly (of human, plant or other complex genomes), human genome phasing, and cancer sequencing.

Reconstructs fragments into long read data
Moleculo technology begins by fragmenting genomic DNA to approximately 10 kilobases. Next, these fragments are clonally amplified, sheared, and marked with a unique barcode. They are then sequenced with Illumina technology. The short sequence reads originating from each molecule are assembled separately. The end result is a full sequence of all the fragments. Essentially, short‐read data is reconstructed into long reads.

Increased throughput, read lengths, and speed
Moleculo technology has the potential to improve the efficiency and accuracy of many existing DNA sequencing applications while increasing the coverage of clinically important genes. These advantages allow for the phased re‐sequencing of human genomes and rapid de novo sequencing of plant and animal genomes.

Sample prep for the Moleculo technology currently takes about 2 days to complete, not including sequencing time. It takes less than 4 days to phase a human genome on the HiSeq 2500, going from sample to answer.

Better genome phasing and de novo sequencing
The long reads produced typically span more than one heterozygous SNP. This allows Moleculo’s proprietary phasing algorithm to "stitch" multiple long reads into a single haplotype and thus phase the genome. The Moleculo technology simplifies de novo sequencing because large repeat regions in the DNA fragments can easily be spanned.

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