Abstract
In this chapter, we describe Long Fragment Read (LFR) technology, a DNA preprocessing method for genome-wide haplotyping by whole genome sequencing (WGS). The addition of LFR prior to WGS on any high-throughput DNA sequencer (e.g., Complete Genomics Revolocity™, BGISEQ-500, Illumina HiSeq, etc.) enables the assignment of single-nucleotide polymorphisms (SNPs) and other genomic variants onto contigs representing contiguous DNA from a single parent (haplotypes) with N50 lengths of up to ~1 Mb. Importantly, this is achieved independent of any parental sequencing data or knowledge of parental haplotypes. Further, the nature of this method allows for the correction of most amplification, sequencing, and mapping errors, resulting in false-positive error rates as low as 10−9. This method can be employed either manually using hand-held micropipettors or in the preferred, automated manner described below, utilizing liquid-handling robots capable of pipetting in the nanoliter range. Automating the method limits the amount of hands-on time and allows significant reduction in reaction volumes. Further, the cost of LFR, as described in this chapter, is moderate, while it adds invaluable whole genome haplotype data to almost any WGS process.
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Acknowledgments
We would like to acknowledge the ongoing contributions and support of all Complete Genomics employees, in particular the many highly skilled individuals who work in the libraries, reagents, and sequencing groups that make it possible to generate high-quality whole genome data. Specifically, we would like to thank Robert Chin, Ramya Srinivasan, Daniel Hayden, and Joseph Peterson for help in improving the conditions and automation processes of this protocol.
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LFR adapter sequences. The sequence information for each of the 384 barcoded adapters is listed in the 5′ to 3′ direction.
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McElwain, M.A., Zhang, R.Y., Drmanac, R., Peters, B.A. (2017). Long Fragment Read (LFR) Technology: Cost-Effective, High-Quality Genome-Wide Molecular Haplotyping. In: Tiemann-Boege, I., Betancourt, A. (eds) Haplotyping. Methods in Molecular Biology, vol 1551. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6750-6_11
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DOI: https://doi.org/10.1007/978-1-4939-6750-6_11
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