Dataset Information

Pilot Screening of Cell-Free mtDNA in NIPT: Quality Control, Variant Calling, and Haplogroup Determination.

ABSTRACT: Clinical tests based on whole-genome sequencing are generally focused on a single task approach, testing one or several parameters, although whole-genome sequencing (WGS) provides us with large data sets that can be used for many supportive analyses. In spite of low genome coverage, data of WGS-based non-invasive prenatal testing (NIPT) contain fully sequenced mitochondrial DNA (mtDNA). This mtDNA can be used for variant calling, ancestry analysis, population studies and other approaches that extend NIPT functionality. In this study, we analyse mtDNA pool from 645 cell-free DNA (cfDNA) samples of pregnant women from different regions of Russia, explore the effects of transportation and storing conditions on mtDNA content, analyse effects, frequency and location of mitochondrial variants called from samples and perform haplogroup analysis, revealing the most common mitochondrial superclades. We have shown that, despite the relatively low sequencing depth of unamplified mtDNA from cfDNA samples, the mtDNA analysis in these samples is still an informative instrument suitable for research and screening purposes.

SUBMITTER: Morshneva A

PROVIDER: S-EPMC8156457 | biostudies-literature | 2021 May

REPOSITORIES: biostudies-literature

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Pilot Screening of Cell-Free mtDNA in NIPT: Quality Control, Variant Calling, and Haplogroup Determination.

Morshneva Alisa A Kozyulina Polina P Vashukova Elena E Tarasenko Olga O Dvoynova Natalia N Chentsova Anastasia A Talantova Olga O Koroteev Alexander A Ivanov Dmitrii D Serebryakova Elena E Ivashchenko Tatyana T Sukhomyasova Aitalina A Maksimova Nadezhda N Bespalova Olesya O Kogan Igor I Baranov Vladislav V Glotov Andrey A

Genes 20210514 5

Clinical tests based on whole-genome sequencing are generally focused on a single task approach, testing one or several parameters, although whole-genome sequencing (WGS) provides us with large data sets that can be used for many supportive analyses. In spite of low genome coverage, data of WGS-based non-invasive prenatal testing (NIPT) contain fully sequenced mitochondrial DNA (mtDNA). This mtDNA can be used for variant calling, ancestry analysis, population studies and other approaches that ex ...[more]

PMID: 34069212

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Project description:BACKGROUND:Although our microbial community and genomes (the human microbiome) outnumber our genome by several orders of magnitude, to what extent the human host genetic complement informs the microbiota composition is not clear. The Human Microbiome Project (HMP) Consortium established a unique population-scale framework with which to characterize the relationship of microbial community structure with their human hosts. A wide variety of taxa and metabolic pathways have been shown to be differentially distributed by virtue of race/ethnicity in the HMP. Given that mtDNA haplogroups are the maternally derived ancestral genomic markers and mitochondria's role as the generator for cellular ATP, characterizing the relationship between human mtDNA genomic variants and microbiome profiles becomes of potential marked biologic and clinical interest. RESULTS:We leveraged sequencing data from the HMP to investigate the association between microbiome community structures with its own host mtDNA variants. 15 haplogroups and 631 mtDNA nucleotide polymorphisms (mean sequencing depth of 280X on the mitochondria genome) from 89 individuals participating in the HMP were accurately identified. 16S rRNA (V3-V5 region) sequencing generated microbiome taxonomy profiles and whole genome shotgun sequencing generated metabolic profiles from various body sites were treated as traits to conduct association analysis between haplogroups and host clinical metadata through linear regression. The mtSNPs of individuals with European haplogroups were associated with microbiome profiles using PLINK quantitative trait associations with permutation and adjusted for multiple comparisons. We observe that among 139 stool and 59 vaginal posterior fornix samples, several haplogroups show significant association with specific microbiota (q-value < 0.05) as well as their aggregate community structure (Chi-square with Monte Carlo, p < 0.005), which confirmed and expanded previous research on the association of race and ethnicity with microbiome profile. Our results further indicate that mtDNA variations may render different microbiome profiles, possibly through an inflammatory response to different levels of reactive oxygen species activity. CONCLUSIONS:These data provide initial evidence for the association between host ancestral genome with the structure of its microbiome.

Project description:BackgroundFor large scale studies aiming at a better understanding of mitochondrial DNA (mtDNA), sequence variation in particular mt haplogroups (hgs) and population structure, reliable low-cost high-throughput genotyping assays are needed. Furthermore, methods facilitating sensitive mixture detection and relative quantification of allele proportions are indispensable for the study of heteroplasmy, mitochondrial sequence evolution, and mitochondrial disorders. Here the properties of a homogeneous competitive duplex allele specific PCR (ARMS) assay were scrutinized in the light of these requirements.Methodology/principal findingsA duplex ARMS assay amplifying either the ancestral mtDNA 2706G allele (non-hg H samples) or the derived 7028C allele (hg H samples) in the presence of SYBR Green fluorescent reporter dye was developed and characterized. Product detection, allele calling, and hg inference were based on the amplicon-characteristic melting-point temperatures obtained with on-line post-PCR fluorescent dissociation curve analysis (DCA). The analytical window of the assay covered at least 5 orders of magnitude of template DNA input with a detection limit in the low picogram range of genomic DNA. A set of forensically relevant test specimens was analyzed successfully. The presence of mtDNA mixtures was detected over a broad range of input DNA amounts and mixture ratios, and the estimation of allele proportions in samples with known total mtDNA content was feasible with limitations. A qualified DNA analyst successfully analyzed approximately 2,200 DNA extracts within three regular working days, without using robotic lab-equipment. By performing the amplification on-line, the assay also facilitated absolute mtDNA quantification.ConclusionsAlthough this assay was developed just for a particular purpose, the approach is general in that it is potentially suitable in a broad variety of assay-layouts for many other applications, including the analysis of mixtures. Homogeneous ARMS-DCA is a valuable tool for large-volume studies targeting small numbers of single nucleotide polymorphisms (SNPs).

Dataset Information

Pilot Screening of Cell-Free mtDNA in NIPT: Quality Control, Variant Calling, and Haplogroup Determination.

Publications

Pilot Screening of Cell-Free mtDNA in NIPT: Quality Control, Variant Calling, and Haplogroup Determination.

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