MHTyper: a microhaplotype allele-calling pipeline for use with next generation sequencing data

Zhang, Chi; Cao, Yandong; Song, Jiao-Jiao; Rao, Min; Nie, Shengjie; Zhang, Guangfeng; Kang, Ke-Lai; Ji, Anquan; Ye, Jian; Wang, Le

doi:10.1080/00450618.2019.1699956

Cited by 7 publications

(3 citation statements)

References 26 publications

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“…The number of MHs in some panels is small, and the detection platform still uses first-generation sequencing [ 16 , 17 , 32 , 40 ]. Moreover, the analysis methods of some MPS panels, such as Flfinder [ 11 ] and MHtyper [ 41 ], are more suitable for their own research analyses. These panels are limited by the number of loci, so the performances of polymorphism, forensic parameters, and mixture detection are limited.…”

Section: Discussionmentioning

confidence: 99%

An MPS-Based 50plex Microhaplotype Assay for Forensic DNA Analysis

Zhang

Xue

Tan

et al. 2023

Genes

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Microhaplotypes (MHs) are widely accepted as powerful markers in forensic studies. They have the advantage of both short tandem repeats (STRs) and single nucleotide polymorphisms (SNPs), with no stutter and amplification bias, short fragments and amplicons, low mutation and recombination rates, and high polymorphisms. In this study, we constructed a panel of 50 MHs that are distributed on 21 chromosomes and analyzed them using the Multiseq multiple polymerase chain reaction (multi-PCR) targeted capture sequencing protocol based on the massively parallel sequencing (MPS) platform. The sizes of markers and amplicons ranged between 11–81 bp and 123–198 bp, respectively. The sensitivity was 0.25 ng, and the calling results were consistent with Sanger sequencing and the Integrative Genomics Viewer (IGV). It showed measurable polymorphism among sequenced 137 Southwest Chinese Han individuals. No significant deviations in the Hardy–Weinberg equilibrium (HWE) and linkage disequilibrium (LD) were found at all MHs after Bonferroni correction. Furthermore, the specificity was 1:40 for simulated two-person mixtures, and the detection rates of highly degraded single samples and mixtures were 100% and 93–100%, respectively. Moreover, animal DNA testing was incomplete and low depth. Overall, our MPS-based 50-plex MH panel is a powerful forensic tool that provides a strong supplement and enhancement for some existing panels.

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Section: Discussionmentioning

confidence: 99%

An MPS-Based 50plex Microhaplotype Assay for Forensic DNA Analysis

Zhang

Xue

Tan

et al. 2023

Genes

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“…Our in‐house pipeline utilized the CIGAR and MD:Z tag information from the BAM files to call variants and determine the locus haplotype of each read. The CIGAR and MD:Z were employed together to extract information on substitutions, insertions, and deletions [30]. We set an analytical threshold of a minimum coverage of 100 reads per locus per sample and 30 reads per haplotype per locus.…”

Section: Methodsmentioning

confidence: 99%

Improving DNA mixtures analysis using compound markers composed of InDels and SNPs screened from the whole genome with next‐generation sequencing

Tan,

Xue,

et al. 2023

Electrophoresis

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Next‐generation sequencing (NGS) allows for better identification of insertion and deletion polymorphisms (InDels) and their combination with adjacent single nucleotide polymorphisms (SNPs) to form compound markers. These markers can improve the polymorphism of microhaplotypes (MHs) within the same length range, and thus, boost the efficiency of DNA mixture analysis. In this study, we screened InDels and SNPs across the whole genome and selected highly polymorphic markers composed of InDels and/or SNPs within 300 bp. Further, we successfully developed and evaluated an NGS‐based panel comprising 55 loci, of which 24 were composed of both SNPs and InDels. Analysis of 124 unrelated Southern Han Chinese revealed an average effective number of alleles (Ae) of 7.52 for this panel. The cumulative power of discrimination and cumulative probability of exclusion values of the 55 loci were 1–2.37 × 10–73 and 1–1.19 × 10–28, respectively. Additionally, this panel exhibited high allele detection rates of over 97% in each of the 21 artificial mixtures involving from two to six contributors at different mixing ratios. We used EuroForMix to calculate the likelihood ratio (LR) and evaluate the evidence strength provided by this panel, and it could assess evidence strength with LR, distinguishing real and noncontributors. In conclusion, our panel holds great potential for detecting and analyzing DNA mixtures in forensic applications, with the capability to enhance routine mixture analysis.

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“…FASTQ data were generated with the Miseq FGx Control Software 1.0.15.0 (Illumina). The MHTyper software 31 was employed for microhaplotype allele calling, with the sequencing depth threshold set at 30 reads. The Hg19 human genome was used as the reference sequence.…”

Section: Multiplex Amplificationmentioning

confidence: 99%

A 124-plex Microhaplotype Panel Based on Next-generation Sequencing Developed for Forensic Applications

Pang

Rao

Chen

et al. 2020

Sci Rep

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Microhaplotypes are an emerging type of forensic genetic marker that are expected to support multiple forensic applications. Here, we developed a 124-plex panel for microhaplotype genotyping based on next-generation sequencing (NGS). The panel yielded intralocus and interlocus balanced sequencing data with a high percentage of effective reads. A full genotype was determined with as little as 0.1 ng of input DNA. Parallel mixture experiments and in-depth comparative analyses were performed with capillary-electrophoresis-based short tandem repeat (STR) and NGS-based microhaplotype genotyping, and demonstrated that microhaplotypes are far superior to STRs for mixture deconvolution. DNA from Han Chinese individuals (n = 256) was sequenced with the 124-plex panel. In total, 514 alleles were observed, and the forensic genetic parameters were calculated. A comparison of the forensic parameters for the 20 microhaplotypes with the top A e values in the 124-plex panel and 20 commonly used forensic STRs showed that these microhaplotypes were as effective as STRs in identifying individuals. A linkage disequilibrium analysis showed that 106 of the 124 microhaplotypes were independently hereditary, and the combined match probability for these 106 microhaplotypes was 5.23 × 10 −66. We conclude that this 124-plex microhaplotype panel is a powerful tool for forensic applications. The microhaplotype is a powerful new type of forensic genetic marker 1,2. It is the combination of two or more closely linked single-nucleotide polymorphisms (SNPs) within DNA segments of 200 base pairs (bp), and offers multiple forensic applications 3-7. Short tandem repeat (STR) genotyping is currently the dominant technology in forensic DNA laboratories. Although it works well with single-sourced DNA samples, great challenges are encountered with DNA mixtures because stutters in the major donor DNA can be indistinguishable from alleles in the minor donor DNA 3,8. Stutters are unavoidable during the replication of repetitive DNA, and they severely interfere with mixture deconvolution. SNPs are not repetitive sequences, but are typically biallelic, which restricts their utility in the analysis of mixtures. Microhaplotypes have the advantages of both STRs and SNPs because they are multiallelic and do not produce stutters during amplification. Therefore, microhaplotypes are perfect genetic markers for mixture deconvolution. Although capillary electrophoresis (CE)-based genetic analyzers are widely used in forensic DNA laboratories, these machines are unsuitable for microhaplotype genotyping 8. Several methods have been used for microhaplotype detection. TaqMan assays have been used to type each SNP that constitutes a microhaplotype 8 , followed by a PHASE software analysis to determine the cis/trans relationships between individual SNP alleles. Single-strand conformational polymorphisms 9 and high-resolution melting curves 4 have also been used for microhaplotype genotyping. These methods are simple and inexpensive, but they can pose problems when multipl...

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MHTyper: a microhaplotype allele-calling pipeline for use with next generation sequencing data

Cited by 7 publications

References 26 publications

An MPS-Based 50plex Microhaplotype Assay for Forensic DNA Analysis

An MPS-Based 50plex Microhaplotype Assay for Forensic DNA Analysis

Improving DNA mixtures analysis using compound markers composed of InDels and SNPs screened from the whole genome with next‐generation sequencing

A 124-plex Microhaplotype Panel Based on Next-generation Sequencing Developed for Forensic Applications

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