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Updated: Jun 24, 2026

Automated Gel Size Selection to Improve the Quality of Next-generation Sequencing Libraries Prepared from Environmental Water Samples
Published on: April 17, 2015
Optimization of 454 sequencing library preparation from small amounts of DNA permits sequence determination of both
Tomislav Maricic1, Svante Pääbo
1Max Planck Institute for Evolutionary Anthropology, Leipzig, Germany. tomislav_maricic@eva.mpg.de
Abstract:
To increase the yield of DNA sequence generated by the 454 technology from small amounts of starting DNA, we investigated the efficiency of each step in the 454 library preparation process. We find that the last step, when the single-stranded library is released by NaOH, is inefficient and highly variable. When this step is replaced with heat treatment, library amounts dramatically increase. Furthermore, when sequencing templates are first isolated by NaOH treatment and subsequently by heat treatment, the sequences of both strands of individual template DNA molecules can be determined. Using this approach, we confirm that C/G base pairs observed as T/A base pairs in Neanderthal DNA sequences are due to a modification of the cytosine rather than guanine residues.
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