Development of High Throughput Process for Constructing 454 Titanium and Illumina Libraries

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We have developed two processes with the Biomek FX robot to construct 454 titanium and Illumina libraries in order to meet the increasing library demands. All modifications in the library construction steps were made to enable the adaptation of the entire processes to work with the 96-well plate format. The key modifications include the shearing of DNA with Covaris E210 and the enzymatic reaction cleaning and fragment size selection with SPRI beads and magnetic plate holders. The construction of 96 Titanium libraries takes about 8 hours from sheared DNA to ssDNA recovery. The processing of 96 Illumina libraries takes less ... continued below

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Deshpande, Shweta; Hack, Christopher; Tang, Eric; Malfatti, Stephanie; Ewing, Aren; Lucas, Susan et al. May 28, 2010.

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We have developed two processes with the Biomek FX robot to construct 454 titanium and Illumina libraries in order to meet the increasing library demands. All modifications in the library construction steps were made to enable the adaptation of the entire processes to work with the 96-well plate format. The key modifications include the shearing of DNA with Covaris E210 and the enzymatic reaction cleaning and fragment size selection with SPRI beads and magnetic plate holders. The construction of 96 Titanium libraries takes about 8 hours from sheared DNA to ssDNA recovery. The processing of 96 Illumina libraries takes less time than that of the Titanium library process. Although both processes still require manual transfer of plates from robot to other work stations such as thermocyclers, these robotic processes represent about 12- to 24-folds increase of library capacity comparing to the manual processes. To enable the sequencing of many libraries in parallel, we have also developed sets of molecular barcodes for both library types. The requirements for the 454 library barcodes include 10 bases, 40-60percent GC, no consecutive same base, and no less than 3 bases difference between barcodes. We have used 96 of the resulted 270 barcodes to construct libraries and pool to test the ability of accurately assigning reads to the right samples. When allowing 1 base error occurred in the 10 base barcodes, we could assign 99.6percent of the total reads and 100percent of them were uniquely assigned. As for the Illumina barcodes, the requirements include 4 bases, balanced GC, and at least 2 bases difference between barcodes. We have begun to assess the ability to assign reads after pooling different number of libraries. We will discuss the progress and the challenges of these scale-up processes.

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  • Sequencing, Finishing and Analysis in the Future

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  • Report No.: LBNL-3670E-Poster
  • Grant Number: DE-AC02-05CH11231
  • DOI: 10.2172/985370 | External Link
  • Office of Scientific & Technical Information Report Number: 985370
  • Archival Resource Key: ark:/67531/metadc1012902

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Office of Scientific & Technical Information Technical Reports

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

Office of Scientific and Technical Information (OSTI) is the Department of Energy (DOE) office that collects, preserves, and disseminates DOE-sponsored research and development (R&D) results that are the outcomes of R&D projects or other funded activities at DOE labs and facilities nationwide and grantees at universities and other institutions.

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  • May 28, 2010

Added to The UNT Digital Library

  • Oct. 14, 2017, 8:36 a.m.

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  • Oct. 17, 2017, 8:10 p.m.

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Deshpande, Shweta; Hack, Christopher; Tang, Eric; Malfatti, Stephanie; Ewing, Aren; Lucas, Susan et al. Development of High Throughput Process for Constructing 454 Titanium and Illumina Libraries, report, May 28, 2010; Berkeley, California. (digital.library.unt.edu/ark:/67531/metadc1012902/: accessed April 23, 2018), University of North Texas Libraries, Digital Library, digital.library.unt.edu; crediting UNT Libraries Government Documents Department.