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United States Department of Agriculture

Agricultural Research Service

Research Project: MOLECULAR GENETIC TOOLS FOR POTATO AND FRUIT TREE IMPROVEMENT Title: Sequencing Higher Eukaryotes from Ultra-Low Starting Concentrations

item Fofanov, Viacheslav -
item McCue, Kent
item Shin, Maria -
item Rojas, Mark -
item Morrell, Jeremy -
item Stevens, Roger -
item Koshinsky, Heather -

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: March 12, 2010
Publication Date: N/A

Technical Abstract: High Throughput Sequencing produces Gigabases of inexpensive sequence data. A limiting factor is the amount of starting material required; Illumina's Paired-End Sample Prep Guide (Feb. 2010) suggests using 1- 5 micrograms of isolated DNA for library preparation. Isolation of this amount of genomic material from clinical, environmental, or forensic samples is not always feasible. We have developed a library preparation protocol that uses 1 microgram isolated genomic DNA. This 1 nanogram, is NOT whole genome amplified, but used directly to generate a library for sequencing. Libraries were prepared with 1 microgram, 10 nanograms, and 1 nanogram of starting Solanum bulbocastanum genomic DNA and used to generate 36 base single-end sequence reads. The results of analyses (by Velvet and internal proprietary sequence analysis tools) were compared and show similar values (Table 1). The proportion of reads mapped to the S. bulbocastanum reference suggests that the starting sample size did not change the distribution of the reads across the reference. As the coverage is less than 1x, the difference in maximum contig length likely reflects random chance and not an underlying bias. Compared to 1 microgram of starting genomic material, library preparation with 1 nanogram of unamplified starting genomic material is feasible and yields sequence reads of similar quality and utility.

Last Modified: 9/4/2015
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