Variant calling and benchmarking in an era of complete human genome sequences

Nature reviews. Genetics

2023 Apr 14

Olson, ND;Wagner, J;Dwarshuis, N;Miga, KH;Sedlazeck, FJ;Salit, M;Zook, JM;
PMID: 37059810 | DOI: 10.1038/s41576-023-00590-0

Genetic variant calling from DNA sequencing has enabled understanding of germline variation in hundreds of thousands of humans. Sequencing technologies and variant-calling methods have advanced rapidly, routinely providing reliable variant calls in most of the human genome. We describe how advances in long reads, deep learning, de novo assembly and pangenomes have expanded access to variant calls in increasingly challenging, repetitive genomic regions, including medically relevant regions, and how new benchmark sets and benchmarking methods illuminate their strengths and limitations. Finally, we explore the possible future of more complete characterization of human genome variation in light of the recent completion of a telomere-to-telomere human genome reference assembly and human pangenomes, and we consider the innovations needed to benchmark their newly accessible repetitive regions and complex variants.

Space in cancer biology: its role and implications

Trends in cancer

2022 Aug 19

Fomitcheva-Khartchenko, A;Kashyap, A;Geiger, T;Kaigala, GV;
PMID: 35995681 | DOI: 10.1016/j.trecan.2022.07.008

Tumor cells present complex behaviors in their interactions with other cells. This intricate behavior is driving the need to develop new tools to understand these ecosystems. The surge of spatial technologies allows evaluation of the complexity of relationships between cells present in a tumor, giving insights about tumor heterogeneity and the tumor microenvironment while providing clinically relevant metrics for tumor classification. In this review, we describe key results obtained using spatial techniques, present recent advances in methods to uncover spatially relevant biological significance, and summarize their main characteristics. We expect spatial technologies to significantly broaden our understanding of tumor biology and to generate clinically relevant tools that will ultimately impact personalized medicine.

Identifying Novel Genes and Variants in Immune and Coagulation Pathways Associated with Macular Degeneration

Ophthalmology Science

2022 Aug 01

Huan, T;Cheng, S;Tian, B;Punzo, C;Lin, H;Daly, M;Seddon, J;
| DOI: 10.1016/j.xops.2022.100206

Purpose To select individuals and families with low genetic burden for age-related macular degeneration (AMD), to inform the clinical diagnosis of macular disorders, and to find novel genetic variants associated with macular disease in affected families. Design Genetic association study based on targeted and whole exome sequencing. Participants 758 subjects (481 individuals with maculopathy and 277 controls), including 316 individuals in 72 families. Methods We focused on 150 genes involved in the complement, coagulation, and inflammatory pathways. Single-variant tests were performed on 3062 variants shared among 5 or more subjects using logistic regression. Gene-based tests were used to evaluate aggregate effects from rare and low frequency variants (at minor allele frequency [MAF]

Allogeneic immunity clears latent virus following allogeneic stem cell transplantation in SIV-infected ART-suppressed macaques

Immunity

2023 May 13

Wu, HL;Busman-Sahay, K;Weber, WC;Waytashek, CM;Boyle, CD;Bateman, KB;Reed, JS;Hwang, JM;Shriver-Munsch, C;Swanson, T;Northrup, M;Armantrout, K;Price, H;Robertson-LeVay, M;Uttke, S;Kumar, MR;Fray, EJ;Taylor-Brill, S;Bondoc, S;Agnor, R;Junell, SL;Legasse, AW;Moats, C;Bochart, RM;Sciurba, J;Bimber, BN;Sullivan, MN;Dozier, B;MacAllister, RP;Hobbs, TR;Martin, LD;Panoskaltsis-Mortari, A;Colgin, LMA;Siliciano, RF;Siliciano, JD;Estes, JD;Smedley, JV;Axthelm, MK;Meyers, G;Maziarz, RT;Burwitz, BJ;Stanton, JJ;Sacha, JB;
PMID: 37236188 | DOI: 10.1016/j.immuni.2023.04.019

Allogeneic hematopoietic stem cell transplantation (alloHSCT) from donors lacking C-C chemokine receptor 5 (CCR5Δ32/Δ32) can cure HIV, yet mechanisms remain speculative. To define how alloHSCT mediates HIV cure, we performed MHC-matched alloHSCT in SIV+, anti-retroviral therapy (ART)-suppressed Mauritian cynomolgus macaques (MCMs) and demonstrated that allogeneic immunity was the major driver of reservoir clearance, occurring first in peripheral blood, then peripheral lymph nodes, and finally in mesenteric lymph nodes draining the gastrointestinal tract. While allogeneic immunity could extirpate the latent viral reservoir and did so in two alloHSCT-recipient MCMs that remained aviremic >2.5 years after stopping ART, in other cases, it was insufficient without protection of engrafting cells afforded by CCR5-deficiency, as CCR5-tropic virus spread to donor CD4+ T cells despite full ART suppression. These data demonstrate the individual contributions of allogeneic immunity and CCR5 deficiency to HIV cure and support defining targets of alloimmunity for curative strategies independent of HSCT.

Histopathologic and proteogenomic heterogeneity reveals features of clear cell renal cell carcinoma aggressiveness

Cancer cell

2022 Dec 20

Li, Y;Lih, TM;Dhanasekaran, SM;Mannan, R;Chen, L;Cieslik, M;Wu, Y;Lu, RJ;Clark, DJ;Kołodziejczak, I;Hong, R;Chen, S;Zhao, Y;Chugh, S;Caravan, W;Naser Al Deen, N;Hosseini, N;Newton, CJ;Krug, K;Xu, Y;Cho, KC;Hu, Y;Zhang, Y;Kumar-Sinha, C;Ma, W;Calinawan, A;Wyczalkowski, MA;Wendl, MC;Wang, Y;Guo, S;Zhang, C;Le, A;Dagar, A;Hopkins, A;Cho, H;Leprevost, FDV;Jing, X;Teo, GC;Liu, W;Reimers, MA;Pachynski, R;Lazar, AJ;Chinnaiyan, AM;Van Tine, BA;Zhang, B;Rodland, KD;Getz, G;Mani, DR;Wang, P;Chen, F;Hostetter, G;Thiagarajan, M;Linehan, WM;Fenyö, D;Jewell, SD;Omenn, GS;Mehra, R;Wiznerowicz, M;Robles, AI;Mesri, M;Hiltke, T;An, E;Rodriguez, H;Chan, DW;Ricketts, CJ;Nesvizhskii, AI;Zhang, H;Ding, L;Clinical Proteomic Tumor Analysis Consortium, ;
PMID: 36563681 | DOI: 10.1016/j.ccell.2022.12.001

Clear cell renal cell carcinomas (ccRCCs) represent ∼75% of RCC cases and account for most RCC-associated deaths. Inter- and intratumoral heterogeneity (ITH) results in varying prognosis and treatment outcomes. To obtain the most comprehensive profile of ccRCC, we perform integrative histopathologic, proteogenomic, and metabolomic analyses on 305 ccRCC tumor segments and 166 paired adjacent normal tissues from 213 cases. Combining histologic and molecular profiles reveals ITH in 90% of ccRCCs, with 50% demonstrating immune signature heterogeneity. High tumor grade, along with BAP1 mutation, genome instability, increased hypermethylation, and a specific protein glycosylation signature define a high-risk disease subset, where UCHL1 expression displays prognostic value. Single-nuclei RNA sequencing of the adverse sarcomatoid and rhabdoid phenotypes uncover gene signatures and potential insights into tumor evolution. In vitro cell line studies confirm the potential of inhibiting identified phosphoproteome targets. This study molecularly stratifies aggressive histopathologic subtypes that may inform more effective treatment strategies.

Accuracy benchmark of the GeneMind GenoLab M sequencing platform for WGS and WES analysis

BMC genomics

2022 Jul 22

Li, C;Fan, X;Guo, X;Liu, Y;Wang, M;Zhao, XC;Wu, P;Yan, Q;Sun, L;
PMID: 35869426 | DOI: 10.1186/s12864-022-08775-3

GenoLab M is a recently developed next-generation sequencing (NGS) platform from GeneMind Biosciences. To establish the performance of GenoLab M, we present the first report to benchmark and compare the WGS and WES sequencing data of the GenoLab M sequencer to NovaSeq 6000 and NextSeq 550 platform in various types of analysis. For WGS, thirty-fold sequencing from Illumina NovaSeq platform and processed by GATK pipeline is currently considered as the golden standard. Thus this dataset is generated as a benchmark reference in this study.GenoLab M showed an average of 94.62% of Q20 percentage for base quality, while the NovaSeq was slightly higher at 96.97%. However, GenoLab M outperformed NovaSeq or NextSeq at a duplication rate, suggesting more usable data after deduplication. For WGS short variant calling, GenoLab M showed significant accuracy improvement over the same depth dataset from NovaSeq, and reached similar accuracy to NovaSeq 33X dataset with 22x depth. For 100X WES, the F-score and Precision in GenoLab M were higher than NovaSeq or NextSeq, especially for InDel calling.GenoLab M is a promising NGS platform for high-performance WGS and WES applications. For WGS, 22X depth in the GenoLab M sequencing platform offers a cost-effective alternative to the current mainstream 33X depth on Illumina.

Data Processing and Germline Variant Calling with the Sentieon Pipeline

Methods in molecular biology (Clifton, N.J.)

2022 Jun 25

Aldana, R;Freed, D;
PMID: 35751805 | DOI: 10.1007/978-1-0716-2293-3_1

Public and private genomic sequencing initiatives generate ever-increasing amounts of genomic data creating a need for improved solutions for genomics data processing (Stephens et al.PLoS Biol 13:e1002195, 2015). The Sentieon Genomics software enables rapid and accurate analysis of next-generation sequence data. In this work, we present a typical use of the Sentieon Genomics software for germline variant calling. The Sentieon germline variant calling pipeline produces more accurate results than other tools on third-party benchmarks (Katherine et al. Front Genet 10:736, 2019; Shen et al. bioRxiv, 885517, 2019) in one tenth the time of comparable pipelines. Parts of this guide come from the official Sentieon Genomics software manual in https://support.sentieon.com/manual (Sentieon. Sentieon Genomics software manual, n.d.) and from the official Sentieon Genomics software application notes in https://support.sentieon.com/appnotes (Sentieon. Sentieon Genomics software application notes, n.d.) and are republished with permission. For additional details and advanced usage instructions of the Sentieon tools, refer to the software manual.

Human microglial models to study HIV infection and neuropathogenesis: a literature overview and comparative analyses

Journal of neurovirology

2022 Feb 09

Gumbs, SBH;Kübler, R;Gharu, L;Schipper, PJ;Borst, AL;Snijders, GJLJ;Ormel, PR;van Berlekom, AB;Wensing, AMJ;de Witte, LD;Nijhuis, M;
PMID: 35138593 | DOI: 10.1007/s13365-021-01049-w

HIV persistence in the CNS despite antiretroviral therapy may cause neurological disorders and poses a critical challenge for HIV cure. Understanding the pathobiology of HIV-infected microglia, the main viral CNS reservoir, is imperative. Here, we provide a comprehensive comparison of human microglial culture models: cultured primary microglia (pMG), microglial cell lines, monocyte-derived microglia (MDMi), stem cell-derived microglia (iPSC-MG), and microglia grown in 3D cerebral organoids (oMG) as potential model systems to advance HIV research on microglia. Functional characterization revealed phagocytic capabilities and responsiveness to LPS across all models. Microglial transcriptome profiles of uncultured pMG showed the highest similarity to cultured pMG and oMG, followed by iPSC-MG and then MDMi. Direct comparison of HIV infection showed a striking difference, with high levels of viral replication in cultured pMG and MDMi and relatively low levels in oMG resembling HIV infection observed in post-mortem biopsies, while the SV40 and HMC3 cell lines did not support HIV infection. Altogether, based on transcriptional similarities to uncultured pMG and susceptibility to HIV infection, MDMi may serve as a first screening tool, whereas oMG, cultured pMG, and iPSC-MG provide more representative microglial culture models for HIV research. The use of current human microglial cell lines (SV40, HMC3) is not recommended.

The assembly of caprine Y chromosome sequence reveals a unique paternal phylogenetic pattern and improves our understanding of the origin of domestic goat

Ecology and evolution

2021 Jun 01

Xiao, C;Li, J;Xie, T;Chen, J;Zhang, S;Elaksher, SH;Jiang, F;Jiang, Y;Zhang, L;Zhang, W;Xiang, Y;Wu, Z;Zhao, S;Du, X;
PMID: 34188851 | DOI: 10.1002/ece3.7611

The mammalian Y chromosome offers a unique perspective on the male reproduction and paternal evolutionary histories. However, further understanding of the Y chromosome biology for most mammals is hindered by the lack of a Y chromosome assembly. This study presents an integrated in silico strategy for identifying and assembling the goat Y-linked scaffolds using existing data. A total of 11.5 Mb Y-linked sequences were clustered into 33 scaffolds, and 187 protein-coding genes were annotated. We also identified high abundance of repetitive elements. A 5.84 Mb subset was further ordered into an assembly with the evidence from the goat radiation hybrid map (RH map). The existing whole-genome resequencing data of 96 goats (worldwide distribution) were utilized to exploit the paternal relationships among bezoars and domestic goats. Goat paternal lineages were clearly divided into two clades (Y1 and Y2), predating the goat domestication. Demographic history analyses indicated that maternal lineages experienced a bottleneck effect around 2,000 YBP (years before present), after which goats belonging to the A haplogroup spread worldwide from the Near East. As opposed to this, paternal lineages experienced a population decline around the 10,000 YBP. The evidence from the Y chromosome suggests that male goats were not affected by the A haplogroup worldwide transmission, which implies sexually unbalanced contribution to the goat trade and population expansion in post-Neolithic period.

Three dimensional models of dedifferentiated liposarcoma cell lines: scaffold-based and scaffold-free approaches

Human cell

2023 Feb 10

Tahara, S;de Faria, FCC;Sarchet, P;Calore, F;Sharick, J;Leight, JL;Casadei, L;Pollock, RE;
PMID: 36763259 | DOI: 10.1007/s13577-023-00865-y

Sarcomas are rare malignancies, the number of reports is limited, and this rarity makes further research difficult even though liposarcoma is one of major sarcomas. 2D cell culture remains an important role in establishing basic tumor biology research, but its various shortcomings and limitations are still of concern, and it is now well-accepted that the behavior of 3D-cultured cells is more reflective of in vivo cellular responses compared to 2D models. This study aimed to establish 3D cell culture of liposarcomas using two different methods: scaffold-based (Matrigel extracellular matrix [ECM] scaffold method) and scaffold-free (Ultra-low attachment [ULA] plate). Lipo246, Lipo224 and Lipo863 cell lines were cultured, and distinctive differences in structures were observed in Matrigel 3D model: Lipo224 and Lipo863 formed spheroids, whereas Lipo246 grew radially without forming spheres. In ULA plate approaches, all cell lines formed spheroids, but Lipo224 and Lipo863 spheroids showed bigger size and looser aggregation than Lipo246. Formalin fixed, paraffin embedded (FFPE) blocks were obtained from all 3D models, confirming the spheroid structures. The expression of MDM2, Ki-67 positivity and MDM2 amplification were confirmed by IHC and DNAscope , respectively. Protein and DNA were extracted from all samples and MDM2 upregulation was confirmed by western blot and qPCR analysis. After treatment with MDM2 inhibitor SAR405838, DDLPS spheroids demonstrated different sensitivity patterns from 2D models. Taken together, we believed that 3D models would have a possibility to provide us a new predictability of efficacy and toxicity, and considered as one important process in in vitro pre-clinical phase prior to moving forward to clinical trials.

The infertile individual analysis based on whole-exome sequencing in chinese multi-ethnic groups

Genes & genomics

2022 Sep 17

Zhao, Q;Li, Y;Liang, Q;Zhao, J;Kang, K;Hou, M;Zhang, X;Du, R;Kong, L;Liang, B;Huang, W;
PMID: 36115009 | DOI: 10.1007/s13258-022-01307-z

Infertility is a common and rapidly growing health issue around the world. The genetic analysis based on the infertile population is crucial for intervention and treatment.To find candidate gene locus led to azoospermia in Chinese multi-ethnic groups and provide theoretical guidance for the diagnosis of genetic diseases to progressively aggravated infertility patients and sterile offspring with ART.The study based on whole-exome sequencing (WES) was presented for genetic characteristic analysis of multi-ethnics and identification of variants related to infertility in Xinjiang area of China.The frequency of pathogenic variants showed significant ethnic differences among four main ethnics in Xinjiang. The population structure analysis confirmed that the Hui was close to the Han population, the Kazak was close to the Uygur population, and there are three ancestry components in the four ethnics. In addition, ten candidate variants potentially regulated azoospermia were detected, and KNTC1 (rs7968222: G > T) was chosen to validate the association. Through the analysis in the valid group, the frequency of rs7968222 (G > T) has a significant difference in the azoospermia population (11.76%, 8/68) and normospermia population (4.63%, 35/756) (P < 0.001). Interestingly, the proportion of people with abnormal follicle-stimulating hormone (FSH) level in the group carrying rs7968222 (G > T) was significantly higher than non-carriers (P < 0.05). Therefore, rs7968222 may regulate spermatogenesis through affecting hormone level.Our study establishes the genetics analysis of Northwest China and finds a candidate gene locus KNTC1 (rs7968222: G > T), which is one of the genetic susceptibility factors for male azoospermia.

Intact HIV proviruses persist in the brain despite viral suppression with ART

Annals of neurology

2022 Jul 22

Cochrane, CR;Angelovich, TA;Byrnes, SJ;Waring, E;Guanizo, AC;Trollope, GS;Zhou, J;Vue, J;Senior, L;Wanicek, E;Jamal Eddine, J;Gartner, MJ;Jenkins, TA;Gorry, PR;Brew, BJ;Lewin, SR;Estes, JD;Roche, M;Churchill, MJ;
PMID: 35867351 | DOI: 10.1002/ana.26456

Human Immunodeficiency Virus (HIV) persistence in blood and tissue reservoirs including the brain is a major barrier to HIV cure and possible cause of comorbid disease. However, the size and replication competent nature of the CNS reservoir is unclear. Here we used the intact proviral DNA assay (IPDA) to provide the first quantitative assessment of the intact and defective HIV reservoir in the brain of people with HIV (PWH).Total, intact and defective HIV proviruses were measured in autopsy frontal lobe tissue from viremic (n=18) or virologically suppressed (n=12) PWH. Total or intact/defective proviruses were measured by detection of HIV pol or the IPDA, respectively, through use of droplet digital PCR (ddPCR). HIV-seronegative individuals were included as controls (n=6).Total HIV DNA was present at similar levels in brain tissues from untreated viremic and antiretroviral (ART)-suppressed individuals (median: 22.3 vs 26.2 HIV pol copies/106 cells), reflecting a stable CNS reservoir of HIV that persists despite therapy. Furthermore, 8/10 viremic and 6/9 virally suppressed PWH also harbored intact proviruses in the CNS (4.63 vs 12.7 intact copies/106 cells). Viral reservoirs in CNS and matched lymphoid tissue were similar in the composition of intact and/or defective proviruses, albeit at lower levels in the brain. Importantly, CNS resident CD68+ myeloid cells in virally suppressed individuals harbored HIV DNA, directly showing the presence of a CNS resident HIV reservoir.Our results demonstrate the first evidence for an intact, potentially replication competent HIV reservoir in the CNS of virally suppressed PWH. This article is protected by

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	Description
sense Example: Hs-LAG3-sense	Standard probes for RNA detection are in antisense. Sense probe is reverse complent to the corresponding antisense probe.
Intron# Example: Mm-Htt-intron2	Probe targets the indicated intron in the target gene, commonly used for pre-mRNA detection
Pool/Pan Example: Hs-CD3-pool (Hs-CD3D, Hs-CD3E, Hs-CD3G)	A mixture of multiple probe sets targeting multiple genes or transcripts
No-XSp Example: Hs-PDGFB-No-XMm	Does not cross detect with the species (Sp)
XSp Example: Rn-Pde9a-XMm	designed to cross detect with the species (Sp)
O# Example: Mm-Islr-O1	Alternative design targeting different regions of the same transcript or isoforms
CDS Example: Hs-SLC31A-CDS	Probe targets the protein-coding sequence only
EnEm	Probe targets exons n and m
En-Em	Probe targets region from exon n to exon m
Retired Nomenclature
tvn Example: Hs-LEPR-tv1	Designed to target transcript variant n
ORF Example: Hs-ACVRL1-ORF	Probe targets open reading frame
UTR Example: Hs-HTT-UTR-C3	Probe targets the untranslated region (non-protein-coding region) only
5UTR Example: Hs-GNRHR-5UTR	Probe targets the 5' untranslated region only
3UTR Example: Rn-Npy1r-3UTR	Probe targets the 3' untranslated region only
Pan Example: Pool	A mixture of multiple probe sets targeting multiple genes or transcripts

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