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Detection of Rare Genomic Variants from Pooled Sequencing Using SPLINTER

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Forensic STR analysis using massive parallel sequencing.

Christophe Van Neste¹, Filip Van Nieuwerburgh, David Van Hoofstat

¹Laboratory of Pharmaceutical Biotechnology, Faculty of Pharmaceutical Sciences, Ghent University, Ghent, Belgium. christophe.vanneste@ugent.be

Forensic Science International. Genetics

|April 17, 2012

Summary

This summary is machine-generated.

Second generation sequencing (SGS) can sequence forensic short tandem repeat (STR) amplicons, but has limitations. An open-source pipeline improves analysis, yielding results comparable to capillary electrophoresis, with added benefits for mixture samples.

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Published on: May 2, 2025

Area of Science:

Forensic Science
Genomics
Molecular Biology

Background:

Short tandem repeat (STR) analysis is crucial in forensic science.
Second generation sequencing (SGS) offers potential advancements in DNA profiling.
Evaluating SGS for forensic STR analysis is essential for technological development.

Purpose of the Study:

To assess the feasibility of using second generation sequencing (SGS) for analyzing multiplexed forensic STR amplicons.
To compare SGS-based STR profiling with traditional capillary electrophoresis methods.
To develop and validate an open-source software pipeline for SGS STR data analysis.

Main Methods:

Utilized Roche GS FLX titanium technology for sequencing multiplexed STR amplicons.
Employed a commercial STR profiling kit (Applied Biosystems AmpFlSTR(®) Profiler Plus(®)).
Developed an open-source software pipeline to process GS FLX FASTA files, including homopolymer compression and allele calling.

Main Results:

Sequencing multiplexed STR amplicons via Roche GS FLX is technically feasible but presents challenges, including a low fraction of full-length reads and high homopolymer error rates.
The developed software pipeline effectively compressed homopolymers and generated comparable qualitative and quantitative results to electrophoresis.
The SGS method identified all alleles in mixed samples where contributors represented at least 10% of the mixture and provided additional information, including allele subtypes.

Conclusions:

Second generation sequencing (SGS) using Roche GS FLX titanium technology is applicable to forensic STR analysis, though not optimal due to read length and homopolymer errors.
The developed open-source pipeline successfully addresses SGS limitations, enabling robust STR profiling comparable to capillary electrophoresis.
SGS offers enhanced capabilities, such as allele subtype identification and improved sensitivity in mixture analysis, making it a valuable tool for forensic investigations.