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RNA-seq03:21

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High-Density DNA and RNA microarrays - Photolithographic Synthesis, Hybridization and Preparation of Large Nucleic Acid Libraries
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An overview of custom array sequencing.

Prachi Kothiyal1, Stephanie Cox, Jonathan Ebert

  • 1Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, USA.

Current Protocols in Human Genetics
|April 11, 2009
PubMed
Summary
This summary is machine-generated.

This guide details oligo hybridization-based resequencing using Affymetrix GeneChip CustomSeq arrays. It covers array design, protocols, data analysis, and indel detection for targeted sequencing applications.

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Area of Science:

  • Genomics
  • Molecular Biology
  • Bioinformatics

Background:

  • Oligo hybridization-based resequencing offers a targeted approach for genetic variation discovery.
  • The Affymetrix GeneChip CustomSeq Resequencing Array platform is a specific implementation of this technology.

Purpose of the Study:

  • To provide a comprehensive overview of oligo hybridization-based resequencing.
  • To detail considerations for implementing the technology and analyzing resulting data.
  • To highlight critical topics in the design, implementation, and analysis of targeted sequencing arrays.

Main Methods:

  • Discussion of array design principles and sequence tiling strategies.
  • Evaluation of experimental protocols including target amplification and PCR.
  • Overview of base-calling algorithms and the Affymetrix GeneChip Sequence Analysis Software (GSEQ).

Main Results:

  • Factors influencing base-calling coverage and accuracy are identified.
  • Performance measures for characterizing base-calling with resequencing arrays are presented.
  • Limitations and approaches for insertion and deletion (indel) detection are discussed.

Conclusions:

  • Successful implementation requires careful consideration of array design, experimental protocols, and data analysis.
  • Understanding factors affecting base-calling accuracy is crucial for reliable resequencing results.
  • Further development is needed for robust indel detection in targeted sequencing.