# Targeted phasing of 2–200 kilobase DNA fragments with a short-read sequencer and a single-tube linked-read library method

## TL;DR

This paper introduces a new method called TELL-Seq that allows scientists to determine which DNA variants come from each parent using short-read sequencing for DNA fragments up to 200 kilobases long.

## Contribution

The novel contribution is a targeted TELL-Seq protocol that enables accurate phasing of 2–200 kb DNA loci using short-read sequencing.

## Key findings

- TELL-Seq can phase DNA fragments up to 200 kb using short-read sequencing.
- The method was validated on clinically relevant genes like BRCA1 and BRCA2.
- Different enrichment methods were successfully used for varying fragment sizes.

## Abstract

In the human genome, heterozygous sites refer to genomic positions with a different allele or nucleotide variant on the maternal and paternal chromosomes. Resolving these allelic differences by chromosomal copy, also known as phasing, is achievable on a short-read sequencer when using a library preparation method that captures long-range genomic information. TELL-Seq is a library preparation that captures long-range genomic information with the aid of molecular identifiers (barcodes). The same barcode is used to tag the reads derived from the same long DNA fragment within a range of up to 200 kilobases (kb), generating linked-reads. This strategy can be used to phase an entire genome. Here, we introduce a TELL-Seq protocol developed for targeted applications, enabling the phasing of enriched loci of varying sizes, purity levels, and heterozygosity. To validate this protocol, we phased 2–200 kb loci enriched with different methods: CRISPR/Cas9-mediated excision coupled with pulse-field electrophoresis for the longest fragments, CRISPR/Cas9-mediated protection from exonuclease digestion for mid-size fragments, and long PCR for the shortest fragments. All selected loci have known clinical relevance: BRCA1, BRCA2, MLH1, MSH2, MSH6, APC, PMS2, SCN5A-SCN10A, and PKI3CA. Collectively, the analyses show that TELL-Seq can accurately phase 2–200 kb targets using a short-read sequencer.