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Targeted Locus Amplification and Haplotyping.

Juliet W Lefferts1,2, Vera Boersma3, Marne C Hagemeijer1,2,4

  • 1Department of Pediatric Respiratory Medicine, Wilhelmina Children's Hospital, University Medical Center Utrecht, Utrecht, The Netherlands.

Methods in Molecular Biology (Clifton, N.J.)
|November 6, 2022
PubMed
Summary
This summary is machine-generated.

Targeted locus amplification (TLA) enables comprehensive genetic variation detection and allelic phasing. This method allows for allele-specific studies of variants within large genomic regions like the CFTR gene.

Keywords:
Cystic fibrosisGenetic variationHaplotypingNext generation sequencingOrganoidsPhasingProximity ligationTargeted Locus Amplification (TLA)

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Area of Science:

  • Genomics
  • Molecular Biology
  • Bioinformatics

Background:

  • Targeted locus amplification (TLA) is a method for detecting genetic variations within specific genomic regions.
  • TLA utilizes proximity ligation to link variants, facilitating allelic phasing and haplotype generation.
  • Understanding genetic variation in an allele-specific manner is crucial for studying genetic diseases.

Purpose of the Study:

  • To provide a detailed protocol for TLA sample preparation.
  • To present a comprehensive bioinformatics pipeline for TLA data analysis, specifically for allelic phasing.
  • To demonstrate TLA's capability in re-sequencing and haplotyping large genes, using the cystic fibrosis transmembrane (CFTR) gene as an example.

Main Methods:

  • Development of a step-by-step protocol for TLA sample preparation.
  • Establishment of a complete bioinformatics pipeline for processing TLA data.
  • Application of TLA to re-sequence and haplotype the entire CFTR gene (>200 kb) from patient-derived intestinal organoids.

Main Results:

  • Successful implementation of the TLA protocol for sample preparation.
  • Validation of the bioinformatics pipeline for accurate allelic phasing of TLA data.
  • Demonstration of TLA's effectiveness in comprehensively analyzing large genes like CFTR, including structural variations and haplotype generation.

Conclusions:

  • TLA is a powerful technique for detecting all genetic variations, including structural variants, within targeted genomic regions.
  • The provided protocol and bioinformatics pipeline enable efficient allelic phasing and haplotype generation from TLA data.
  • TLA is suitable for in-depth analysis of large genes and can be applied to patient-derived samples for genetic studies.