Large Gap Size Paired-end Library Construction for Second Generation Sequencing

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Fosmid or BAC end sequencing plays an important role in de novo assembly of large genomes like fungi and plants. However construction and Sanger sequencing of fosmid or BAC libraries are laborious and costly. The current 454 Paired-End (PE) Library and Illumina Jumping Library construction protocols are limited with the gap sizes of approximately 20 kb and 8 kb, respectively. In the attempt to understand the limitations of constructing PE libraries with greater than 30Kb gaps, we have purified 18, 28, 45, and 65Kb sheared DNA fragments from yeast and circularized the ends using the Cre-loxP approach described in the ... continued below

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Peng, Ze; Hamilton, Matthew; Froula, Jeff; Ewing, Aren; Foster, Brian & Cheng, Jan-Fang May 28, 2010.

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Fosmid or BAC end sequencing plays an important role in de novo assembly of large genomes like fungi and plants. However construction and Sanger sequencing of fosmid or BAC libraries are laborious and costly. The current 454 Paired-End (PE) Library and Illumina Jumping Library construction protocols are limited with the gap sizes of approximately 20 kb and 8 kb, respectively. In the attempt to understand the limitations of constructing PE libraries with greater than 30Kb gaps, we have purified 18, 28, 45, and 65Kb sheared DNA fragments from yeast and circularized the ends using the Cre-loxP approach described in the 454 PE Library protocol. With the increasing fragment sizes, we found a general trend of decreasing library quality in several areas. First, redundant reads and reads containing multiple loxP linkers increase when the average fragment size increases. Second, the contamination of short distance pairs (<10Kb) increases as the fragment size increases. Third, chimeric rate increases with the increasing fragment sizes. We have modified several steps to improve the quality of the long span PE libraries. The modification includes (1) the use of special PFGE program to reduce small fragment contamination; (2) the increase of DNA samples in the circularization step and prior to the PCR to reduce redundant reads; and (3) the decrease of fragment size in the double SPRI size selection to get a higher frequency of LoxP linker containing reads. With these modifications we have generated large gap size PE libraries with a much better quality.

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  • Sequencing, Finishing and Analysis in the Future

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  • Report No.: LBNL-3668E-Poster
  • Grant Number: DE-AC02-05CH11231
  • DOI: 10.2172/985369 | External Link
  • Office of Scientific & Technical Information Report Number: 985369
  • Archival Resource Key: ark:/67531/metadc1014672

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Reports, articles and other documents harvested from the Office of Scientific and Technical Information.

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  • May 28, 2010

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  • Oct. 14, 2017, 8:36 a.m.

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  • Oct. 18, 2017, 10:09 a.m.

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Peng, Ze; Hamilton, Matthew; Froula, Jeff; Ewing, Aren; Foster, Brian & Cheng, Jan-Fang. Large Gap Size Paired-end Library Construction for Second Generation Sequencing, report, May 28, 2010; Berkeley, California. (digital.library.unt.edu/ark:/67531/metadc1014672/: accessed June 19, 2018), University of North Texas Libraries, Digital Library, digital.library.unt.edu; crediting UNT Libraries Government Documents Department.