Haplotracker: a web application for simple and accurate mitochondrial haplogrouping using short DNA fragments

Abstract

Mitochondrial DNA (mtDNA) haplogrouping is widely used in population genetics, forensics, and medical research to study evolutionary questions in human populations, examine degraded remains, and search for mtDNA-associated diseases. Next-generation sequencing methods have become a revolutionary tool for mtDNA analysis, particularly for degraded DNA, but they remain costly, laborious, and time-consuming. If an accurate and simple haplogroup-tracking tool were available, haplogrouping could be performed easily and rapidly. Here, we present Haplotracker, a web application for highly accurate and simple haplogroup tracking using a few sequence fragments. Haplotracker offers a unique user-friendly interface and estimates highly probable haplogroups from control-region sequences using a novel algorithm based on Phylotree Build 17 and our haplotype database (n=118,869). Haplotracker provides a simple, novel HG tracking solution, which was established through repeated blind simulation tests. It narrows down potential haplogroups and identifies their differential coding-region variants to confirm the haplogroups or to track sub-haplogroups. Haplotracker gives detailed information on sample variants, including their frequency in a large mtGenome database, which may give researchers an insight into common, rare, and potentially pathogenic mutations. It also offers a conserved region mapping tool for PCR primer design for successful tracking. Haplotracker produced high haplogroup prediction accuracy using 8,216 control-region Phylotree-provided sequences. It estimated top-ranking haplogroups with a higher concordance rate (56.6%, p<0.0001) than the similar tools MitoTool (29.4%) and HaploGrep 2 (33.9%). The significance persisted up to Rank 30. Haplotracker accurately estimated super-HGs from 94% of the control-region sequences at Rank 1. Further evaluation of the accuracy with 46,322 control-region sequences was significant. Laboratory application of Haplotracker to an ancient DNA extract demonstrated its practical usefulness. These results highlight the potential for the use of our web application as an alternative to full genome sequencing for easy haplogrouping, which may be useful in related fields. Free access: https://haplotracker.cau.ac.kr. Author summaryMitochondrial haplogroup (HG) classification is required in the search for answers to evolutionary questions about human populations, in the investigation of forensic samples, and in the search for disease-associated mutations. The sequencing of mitochondrial DNA (mtDNA) at the genome level is now frequently used due to the falling cost of next-generation sequencing. It offers accurate and detailed analysis of mtDNA variation, but it is still costly, laborious, and time-consuming. If an accurate and simple HG tracking tool were available, haplogrouping could be performed easily and rapidly. Here, we developed Haplotracker, a simple and accurate HG tracking web application with a novel algorithm and a novel tracking tool. The highly accurate prediction performance of Haplotracker was demonstrated in a series of tests. Using only control-region sequences, it accurately predicted HGs in more than half of the total Phylotree-provided mtGenome sequences and approximately 80% up to Rank 5; for super-HGs, it accurately predicted 94% at Rank 1 and more than 98% up to Rank 3. We demonstrated simple tracking for HG confirmation using Haplotracker. These results show that mtDNA haplogrouping with our web application may be useful in related fields.