Targeted Phasing of 2-200 Kilobase DNA Fragments with a Short-Read Sequencer and a Single-Tube Linked-Read Library...

Area of Science:

• Genomics
• Molecular Biology
• Bioinformatics

Background:

• Phasing heterozygous sites in the human genome is crucial for understanding genetic variation.
• Existing linked-reads technologies like TELL-Seq are effective for whole-genome phasing but not cost-efficient for targeted applications.
• Short-read sequencing offers high accuracy and low cost but typically lacks long-range information for phasing.

Approach:

• Developed an optimized Targeted TELL-Seq protocol for cost-effective phasing of specific genomic loci.
• Utilized CRISPR/Cas9 for enrichment of target regions (2-200 kb) prior to library preparation.
• Incorporated unique molecular identifiers (barcodes) to generate linked-reads from high-molecular-weight DNA fragments.

Key Points:

• Targeted TELL-Seq maximizes linked-read efficiency and library yield while minimizing input DNA and sequencing costs.
• The protocol successfully phased variants in clinically relevant genes including BRCA1, BRCA2, MLH1, and SCN5A.
• Demonstrated reliable phasing of target loci ranging from 2 kb to 200 kb.

Conclusions:

• Targeted TELL-Seq provides a cost-effective and accurate method for phasing allelic variants within specific genomic regions.
• This approach bridges the gap between the accuracy of short-read sequencing and the long-range information needed for phasing.
• Enables targeted genetic analysis with the benefits of linked-reads technology.