Targeted Locus Amplification and Haplotyping

Juliet W Lefferts; Vera Boersma; Marne C Hagemeijer; Karima Hajo; Jeffrey M Beekman; Erik Splinter

doi:10.1007/978-1-0716-2819-5_2

Targeted Locus Amplification and Haplotyping

Methods Mol Biol. 2023:2590:31-48. doi: 10.1007/978-1-0716-2819-5_2.

Authors

Juliet W Lefferts^#^{1

2}, Vera Boersma^#³, Marne C Hagemeijer^{1

2

4}, Karima Hajo³, Jeffrey M Beekman^{1

2}, Erik Splinter⁵

Affiliations

¹ Department of Pediatric Respiratory Medicine, Wilhelmina Children's Hospital, University Medical Center Utrecht, Utrecht, The Netherlands.
² Regenerative Medicine Center, Center for Living Technologies, University Medical Center Utrecht, Utrecht, The Netherlands.
³ Cergentis BV, Utrecht, The Netherlands.
⁴ Department of Clinical Genetics, Center for Lysosomal and Metabolic Diseases, Erasmus University Medical Center, Rotterdam, The Netherlands.
⁵ Cergentis BV, Utrecht, The Netherlands. erik.splinter@cergentis.com.

^# Contributed equally.

PMID: 36335490
DOI: 10.1007/978-1-0716-2819-5_2

Abstract

Targeted locus amplification (TLA) allows for the detection of all genetic variation (including structural variation) in a genomic region of interest. As TLA is based on proximity ligation, variants can be linked to each other, thereby enabling allelic phasing and the generation of haplotypes. This allows for the study of genetic variants in an allele-specific manner. Here, we provide a step-by-step protocol for TLA sample preparation and a complete bioinformatics pipeline for the allelic phasing of TLA data. Additionally, to illustrate the protocol, we show the ability of TLA to re-sequence and haplotype the complete cystic fibrosis transmembrane (CFTR) gene (> 200 kb in size) from patient-derived intestinal organoids.

Keywords: Cystic fibrosis; Genetic variation; Haplotyping; Next generation sequencing; Organoids; Phasing; Proximity ligation; Targeted Locus Amplification (TLA).

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Alleles
Cystic Fibrosis* / genetics
Genomics* / methods
Haplotypes / genetics
Humans