Whole-genome Sequencing Reads Research Articles

Reproducibility of next-generation-sequencing-based analysis of a CRISPR/Cas9 genome edited oil seed rape

Next-generation-sequencing (NGS) becomes increasingly important for laboratories tasked with the detection of genetically modified organisms (GMOs) in food, feed and seeds. Its implementation into standardized workflows demands reliable intra- and inter-laboratory reproducibility. Here, we analyze the reproducibility of short- and long-read targeted NGS and long-read whole genome sequencing (WGS) data between three independent laboratories. Replicate samples were submitted for sequencing and comparatively analyzed. The targeted-NGS-samples consisted of oil seed rape (OSR) sampled from a commodity shipment spiked with a genome edited (GE) OSR and the WGS-samples consisted of leaf material from the GMOs’ parental line. All laboratories delivered highly reproducible high-quality targeted NGS data with little variation. The detection of GMO-related sequences works well regardless of the facility, while the mapping to the complex genome is superior using long read data. Long read WGS is currently not suitable for routine use in enforcement laboratories, due to a large inter-laboratory variation.

A comparative investigation of single nucleotide variant calling for a personal non-Caucasian sequencing sample.

Dropping cost and increasing clinical application of whole genome sequencing (WGS) lead a necessity of efficient (accurate and rapid) variant calling procedures from a personal WGS data (n = 1). A number of variant calling pipelines have been introduced utilizing the human genome reference GRCh38 as a reference and a benchmark dataset called 'NA12878', which are both 'standard' but limited ethnic origin. Considering the nature of variant calling algorithms and recent updates in sequencing protocol, however, it is necessary to revisit the efficiency of the current best pipelines for a personal WGS data from diverse ethnicity. We discuss the most efficient practices for variant calling of a personal WGS reads, with a particular emphasis on whether (1) ethnic match or mismatch between the reference genome and a WGS data produces a distinct result and more importantly (2) there is an ethnic-specific optimal workflow. Here, we generate an appropriate WGS data, DNA array, and sufficient number of Sanger validated variants from a single Korean subject to perform such a comprehensive comparison. We applied this WGS reads and the 'NA12878' reads to 8 different variant calling pipelines with 2 different reference genomes (GRCh38 and KOREF, a Korean reference genome) to which the WGS reads from different ethnic origins are aligned. We evaluated the performance of the pipelines with the matched array genotype data and Sanger sequencing validation and demonstrated that: regardless to the ethnic match/mismatch (1) Novoalign-GATK4 showed the most efficient performance with the exceptional calls in MHC region; (2) the overall performance was better with GRCh38, while a significant difference in recall was observed. In addition, we found it is largely reduced computing cost maintaining performance to remove 'markduplication' step with PCR-free WGS data. For variant calling of a personal PCR-free WGS data, regardless of ethnicity consideration, we recommend the use of the Novoalign + GATK4 with GRCh38 and without 'markduplication'.

The Good, The Bad, and The Misleading: How to Improve the Quality of 'Genome Announcements'?

When comparing the requirements of diverse journals to publish microbial 'Genome Reports' we noticed that some mostly focus on Benchmarking Universal Single-Copy Orthologs (BUSCO) scores as a quality measure while the exclusion of possible contaminating sequences from genomic resources and the possible mis-identification of the target microbes receive less attention. To deal with these quality issues, we suggest that DNA barcodes that are widely accepted for the identification of the target microbe species should be extracted from newly reported genome resources and included in phylogenetic analyses to confirm the identity of the sequenced microorganisms before 'Genome Reports' are published. This approach, applied, for example, by the journal IMA Fungus, largely prevents the mis-identification of the microbes that are targeted for whole genome sequencing (WGS). In addition, contig similarity values, including GC-content, remapping coverage of WGS reads, and BLASTN searches against the NCBI nt database, will also reveal contamination issues. The values of these two recommendations to improve the publication criteria for microbial 'Genome Reports' in diverse journals are demonstrated here through analyses of a draft genome published in Molecular Plant-Microbe Interactions, then retracted due to contaminations.

Analysis of coverage of Alu repeats by aligned genomic reads

Alu repeats occupy a notable part of the human genome and greatly affect processes related to genome integrity maintenance. One of the basic methods for studying variation in a genome, including Alu repeats is genome sequencing followed by mapping the sequenced reads to a reference genome sequence. The key feature of the read alignment is the depth of reference genome region coverage by mapped reads. In this paper, a new method is proposed for analyzing the coverage of Alu repeats and their flanking regions by whole-genome sequencing reads and the distribution of mean coverage in two aforementioned region types is explored.

Adequacy of using a single nasal swab for rapid influenza diagnostic testing, PCR, and whole genome sequencing.

Rapid influenza diagnostic tests (RIDT) demonstrate varying sensitivities, often necessitating reverse transcriptase polymerase chain reaction (RT-PCR) to confirm results. The two methods generally require separate specimens. Using the same anterior nasal swab for both RIDT and molecular confirmation would reduce cost and waste and increase patient comfort. The aim of this study was to determine if RIDT residual nasal swab (rNS) specimens are adequate for RT-PCR and whole genome sequencing (WGS). We performed RT-PCR and WGS on paired rNS and nasopharyngeal or oropharyngeal (NP/OP) swab specimens that were collected from primary care patients across all ages. We randomly selected 199 and 40 paired specimens for RT-PCR and WGS, respectively, from the 962 paired surveillance specimens collected during the 2014-2015 influenza season. Sensitivity and specificity for rNS specimens were 81.3% and 96.7%, respectively, as compared to NP/OP specimens. The mean cycle threshold (Ct) value for the NP/OP specimen was significantly lower when the paired specimens were both positive than when the NP/OP swab was positive and the nasal swab was negative (25.5 vs 29.5; p<0.001). Genomic information was extracted from all 40 rNS specimens and 37 of the 40 NP/OP specimens. Complete WGS reads were available for 67.5% (14 influenza A; 13 influenza B) of the rNS specimens and 59.5% (14 influenza A; 8 influenza B) of the NP/OP specimens. It is feasible to use a single anterior nasal swab for RIDT followed by RT-PCR and/or WGS. This approach may be appropriate in situations where training and supplies are limited. Additional studies are needed to determine if residual nasal swabs from other rapid diagnostic tests produce similar results.

Transmission Dynamics of a Mycobacterium tuberculosis Complex Outbreak in an Indigenous Population in the Colombian Amazon Region.

Whole genome sequencing (WGS) has become the main tool for studying the transmission of Mycobacterium tuberculosis complex (MTBC) strains; however, the clonal expansion of one strain often limits its application in local MTBC outbreaks. The use of an alternative reference genome and the inclusion of repetitive regions in the analysis could potentially increase the resolution, but the added value has not yet been defined. Here, we leveraged short and long WGS read data of a previously reported MTBC outbreak in the Colombian Amazon Region to analyze possible transmission chains among 74 patients in the indigenous setting of Puerto Nariño (March to October 2016). In total, 90.5% (67/74) of the patients were infected with one distinct MTBC strain belonging to lineage 4.3.3. Employing a reference genome from an outbreak strain and highly confident single nucleotide polymorphisms (SNPs) in repetitive genomic regions, e.g., the proline-glutamic acid/proline-proline-glutamic-acid (PE/PPE) gene family, increased the phylogenetic resolution compared to a classical H37Rv reference mapping approach. Specifically, the number of differentiating SNPs increased from 890 to 1,094, which resulted in a more granular transmission network as judged by an increasing number of individual nodes in a maximum parsimony tree, i.e., 5 versus 9 nodes. We also found in 29.9% (20/67) of the outbreak isolates, heterogenous alleles at phylogenetically informative sites, suggesting that these patients are infected with more than one clone. In conclusion, customized SNP calling thresholds and employment of a local reference genome for a mapping approach can improve the phylogenetic resolution in highly clonal MTBC populations and help elucidate within-host MTBC diversity. IMPORTANCE The Colombian Amazon around Puerto Nariño has a high tuberculosis burden with a prevalence of 1,267/100,000 people in 2016. Recently, an outbreak of Mycobacterium tuberculosis complex (MTBC) bacteria among the indigenous populations was identified with classical MTBC genotyping methods. Here, we employed a whole-genome sequencing-based outbreak investigation in order to improve the phylogenetic resolution and gain new insights into the transmission dynamics in this remote Colombian Amazon Region. The inclusion of well-supported single nucleotide polymorphisms in repetitive regions and a de novo-assembled local reference genome provided a more granular picture of the circulating outbreak strain and revealed new transmission chains. Multiple patients from different settlements were possibly infected with at least two different clones in this high-incidence setting. Thus, our results have the potential to improve molecular surveillance studies in other high-burden settings, especially regions with few clonal multidrug-resistant (MDR) MTBC lineages/clades.

ONT long-read WGS for variant discovery and orthogonal confirmation of short read WGS derived genetic variants in clinical genetic testing.

Technological advances in Next-Generation Sequencing dramatically increased clinical efficiency of genetic testing, allowing detection of a wide variety of variants, from single nucleotide events to large structural aberrations. Whole Genome Sequencing (WGS) has allowed exploration of areas of the genome that might not have been targeted by other approaches, such as intergenic regions. A single technique detecting all genetic variants at once is intended to expedite the diagnostic process while making it more comprehensive and efficient. Nevertheless, there are still several shortcomings that cannot be effectively addressed by short read sequencing, such as determination of the precise size of short tandem repeat (STR) expansions, phasing of potentially compound recessive variants, resolution of some structural variants and exact determination of their boundaries, etc. Therefore, in some cases variants can only be tentatively detected by short reads sequencing and require orthogonal confirmation, particularly for clinical reporting purposes. Moreover, certain regulatory authorities, for example, New York state CLIA, require orthogonal confirmation of every reportable variant. Such orthogonal confirmations often involve numerous different techniques, not necessarily available in the same laboratory and not always performed in an expedited manner, thus negating the advantages of "one-technique-for-all" approach, and making the process lengthy, prone to logistical and analytical faults, and financially inefficient. Fortunately, those weak spots of short read sequencing can be compensated by long read technology that have comparable or better detection of some types of variants while lacking the mentioned above limitations of short read sequencing. At Variantyx we have developed an integrated clinical genetic testing approach, augmenting short read WGS-based variant detection with Oxford Nanopore Technologies (ONT) long read sequencing, providing simultaneous orthogonal confirmation of all types of variants with the additional benefit of improved identification of exact size and position of the detected aberrations. The validation study of this augmented test has demonstrated that Oxford Nanopore Technologies sequencing can efficiently verify multiple types of reportable variants, thus ensuring highly reliable detection and a quick turnaround time for WGS-based clinical genetic testing.

Molecular Epidemiology of Global Carbapenemase-Producing Citrobacter spp. (2015-2017).

The emergence of carbapenem resistance is a significant public health concern. The rate of infections caused by carbapenemase-producing Citrobacter spp., particularly C. freundii, is increasing. Concomitantly, comprehensive global genomic data on carbapenemase-producing Citrobacter spp. are scarce. We used short read whole-genome sequencing to describe the molecular epidemiology and international distribution of eighty-six carbapenemase-producing Citrobacter spp. obtained from two surveillance programs (2015 to 17). The common carbapenemases were KPC-2 (26%), VIM-1 (17%), IMP-4 (14%) and NDM-1 (10%). C. freundii and C. portucalensis were the principal species. C. freundii consisted of multiple clones obtained mainly from Colombia (with KPC-2), the United States (with KPC-2, -3), and Italy (with VIM-1). Two dominant C. freundii clones were identified: ST98 was linked with blaIMP-8 from Taiwan and blaKPC-2 from the United States, and ST22 was linked with blaKPC-2 from Colombia and blaVIM-1 from Italy. C. portucalensis consisted mainly of two clones: ST493 with blaIMP-4 which was limited to Australia, and ST545 with blaVIM-31 which was limited to Turkey. Class I integron (In916) with blaVIM-1 was circulating between multiple sequence types (STs) in Italy, Poland, and Portugal. In73 with blaIMP-8 was circulating between various STs in Taiwan, while In809 with blaIMP-4 was circulating between different STs in Australia. The global carbapenemase-producing Citrobacter spp. population is dominated by diverse STs with different characteristics and varied geographical distribution and thus requires continued monitoring. Ongoing genomic surveillance should use methodologies able to distinguish between C. freundii and C. portucalensis. IMPORTANCE Citrobacter spp. are gaining recognition as important causes of hospital-acquired infections in humans. Among Citrobacter spp., carbapenemase-producing strains are cause of utmost concern to health care services globally due to their ability to resist therapy with virtually any beta-lactam antibiotic. Here, we described the molecular characteristics of a global collection of carbapenemase-producing Citrobacter spp. C. freundii and C. portucalensis were the most common species among Citrobacter spp. with carbapenemases from this survey. Importantly, C. portucalensis was misidentified as C. freundii when using Vitek 2.0/MALDI-TOF MS (matrix-assisted laser desorption/ionization-time of flight mass spectrometry) phenotypic identification, which has important implications for future surveys. Among C. freundii, we identified two dominant clones: ST98 with blaIMP-8 from Taiwan and blaKPC-2 from the United States, and ST22 with blaKPC-2 from Colombia and blaVIM-1 from Italy. As for C. portucalensis, the dominant clones consisted of ST493 with blaIMP-4 from Australia and ST545 with blaVIM-31 from Turkey.

GAMBIT (Genomic Approximation Method for Bacterial Identification and Tracking): A methodology to rapidly leverage whole genome sequencing of bacterial isolates for clinical identification.

Whole genome sequencing (WGS) of clinical bacterial isolates has the potential to transform the fields of diagnostics and public health. To realize this potential, bioinformatic software that reports identification results needs to be developed that meets the quality standards of a diagnostic test. We developed GAMBIT (Genomic Approximation Method for Bacterial Identification and Tracking) using k-mer based strategies for identification of bacteria based on WGS reads. GAMBIT incorporates this algorithm with a highly curated searchable database of 48,224 genomes. Herein, we describe validation of the scoring methodology, parameter robustness, establishment of confidence thresholds and the curation of the reference database. We assessed GAMBIT by way of validation studies when it was deployed as a laboratory-developed test in two public health laboratories. This method greatly reduces or eliminates false identifications which are often detrimental in a clinical setting.

A reference genome for Bluegill (Centrarchidae: Lepomis macrochirus).

North American sunfishes (Family Centrarchidae) are among the most popular sportfish throughout the United States and Canada. Despite the popularity of sunfishes, their ecological importance, and their extensive stocking and aquacultural history, few molecular studies have examined the evolutionary relationships and species boundaries among members of this group, many of which are known to hybridize. Here, we describe a chromosome-scale genome assembly representing Bluegill (Lepomis macrochirus), one of the most widespread centrarchid species. By combining long-read, Oxford Nanopore sequencing data with short-insert, whole-genome and HiC sequence reads, we produced an assembly (Lm_LA_1.1) having a total length of 889 Mb including 1,841 scaffolds and having a scaffold N50 of 36 Mb, L50 of 12, N90 of 29 Mb, and L90 of 22. We detected 99% (eukaryota_odb10) and 98% (actinopterygii_odb10) universal single-copy orthologs (BUSCOs), and ab initio gene prediction performed using this new assembly identified a set of 17,233 genes that were supported by external (OrthoDB v10) data. This new assembly provides an important addition to the growing set of assemblies already available for spiny-rayed fishes (Acanthomorpha), and it will serve as a resource for future studies that focus on the complex evolutionary history of centrarchids.

Characterization of the immunoglobulin lambda chain locus from diverse populations reveals extensive genetic variation.

Immunoglobulins (IGs), crucial components of the adaptive immune system, are encoded by three genomic loci. However, the complexity of the IG loci severely limits the effective use of short read sequencing, limiting our knowledge of population diversity in these loci. We leveraged existing long read whole-genome sequencing (WGS) data, fosmid technology, and IG targeted single-molecule, real-time (SMRT) long-read sequencing (IG-Cap) to create haplotype-resolved assemblies of the IG Lambda (IGL) locus from 6 ethnically diverse individuals. In addition, we generated 10 diploid assemblies of IGL from a diverse cohort of individuals utilizing IG-Cap. From these 16 individuals, we identified significant allelic diversity, including 36 novel IGLV alleles. In addition, we observed highly elevated single nucleotide variation (SNV) in IGLV genes relative to IGL intergenic and genomic background SNV density. By comparing SNV calls between our high quality assemblies and existing short read datasets from the same individuals, we show a high propensity for false-positives in the short read datasets. Finally, for the first time, we nucleotide-resolved common 5-10 Kb duplications in the IGLC region that contain functional IGLJ and IGLC genes. Together these data represent a significant advancement in our understanding of genetic variation and population diversity in the IGL locus.

Diagnostic Utility of Whole Genome Sequencing in Adults with B-Other Acute Lymphoblastic Leukaemia

Background B-other ALL represents a working definition for patients with B cell precursor (BCP) ALL without a known primary chromosomal abnormality at diagnosis. In this study we use whole genome sequencing (WGS) to characterize the molecular pathogenesis of adult B-other cases (age ≥ 25yrs) from the UKALL14 trial (NCT01085617). Methods Of 652 patients aged 25-65yrs enrolled onto UKALL14, 333 (51%) had B-other ALL. Sufficient material was available to screen 156/333 B-other cases for recurrent Ph-like fusion events (CRLF2, JAK2, ABL1, ABL2 & PDGFRB) using FISH and MLPA (kit P335). This identified 28 (18%) "Ph-like" fusion events (21 CRLF2, 5 ABL-class and 2 JAK2). Of the remaining 128 B-other cases 57 had available samples for tumor normal paired WGS (read depth 60x and 30x respectively). Diagnostic RNA was available for 33 of the 57 cases Bioinformatic analysis was performed to determine small somatic mutations; single nucleotide variants (SNVs) and insertion/deletions (INDELs) as well as copy number aberrations (CNA) and structural variants (SV). We developed WGS approaches to identify IGH enhancer hijack and DUX4 rearrangements, utilizing gGnome and GRIDSS respectively. For 33 cases RNA-seq data was analyzed to validate fusion genes and genetic classification into one of 18 ALL subtypes using the ALLSorts v2 classifier. Results 52/57 cases passed QC. In 5 (10%) cases an established WHO2016 genetic subtype was identified that was missed by standard of care including three high-risk (2 low hypodiploid and a near haploid) and 2 standard risk (high hyperdiploid and TCF3::PBX1) cases. Cytogenetic analysis for these 5 cases either failed (n=2) or produced a normal karyotype (n=3). The WGS inferred tumor purity for these 5 cases was variable with a median of 46% and a range of 33% to 96% compared to 89% (range 13%-99%) for the remaining cases (n=47) in the dataset. Among the remaining 47 B-other cases, WGS analysis identified abnormalities enabling 41 (87%) cases to be classified into a genetic ALL subtype: DUX4 rearrangements (DUX4r, n=8), PAX5alt (n=7), PAX5 P80R (n=4), ZNF384 rearrangements (ZNF384r, n=5), ZEB2/CEBP (n=4), MEF2D rearrangements (MEF2Dr, n=3), UBTF::ATXN7L3 fusions (n=2), IDH1/2 mutations (n=2), JAK-STAT abnormalities (n=2), along with single cases of IGK::BCL2, IGH::CEBPA, IGH::ID4 and IGH::MIR125B1. WGS resolves 8 cases with complex karyotype into one of 6 different genomic subtypes: 2xDUX4r, 2xMEF2Dr, ZEB2/CEBP, UBTF::ATXN7L3, IGK::BCL2 & IGH::ID4. The MEF2Dr subtype (n=3), is enriched in chromothripsis (2/3), and complex SVs targeting the MEF2D gene. Lastly, in all cases with PAX5 mutations (PAX5 P80R (n=4) and PAX5alt (n=4)) we observe bi-allelic targeting of locus by mutations, deletions or LOH. The remaining 6 (13%) B-other cases could not be classified into a recognized B-other subtype. A MYO18A::FGFR1 fusion (t(8;17)(p11;q23)), which most likely maps to the WHO2016 "Lymphoid neoplasms with FGFR1 rearrangement" classification. Two cases had clonal frameshifts in genes associated with T-ALL (PTPN2 p.A108fs*5 and WT1 p.V371fs*14 respectively). A case with a somatic ETV6 p.R399C previously reported in germline risk with a ZNF292::KDM6A fusion. Lastly a case with a clonal mutation typically associated with myeloid disease ASXL1 p.R417*. Among the 52 cases studied, WGS only failed to identify a driver or putative driver in one case, which had the lowest burden of SNVs (861) and an estimated tumor purity, 28%. For the 31 cases with both DNA and RNA, WGS and RNA analysis were concordant for 100% of cases, with RNA identifying an additional KDM6A::CGA fusion. In 3 cases WGS provides evidence that would have challenged diagnosis and/or treatment; IGK::BCL2 with BCL2 and MYC overexpression, IGH::DUX4 with hypermutation (MSH6 loss) and IGH::ID4 with homozygous loss of CD58 and concurrent LOH of HLA-B. Conclusions WGS assigned 88% (46/52) of cases called B-other to an established genetic subtype of ALL. This includes 5 cases with WHO2016 defined subtypes, 3 were high-risk ploidy subtypes that would have changed their UKALL14 risk group and post-induction treatment in the absence of other risk factors. Among the true B-other cases, 87% (41/47) were assigned to one of the newly described genetic subtypes of ALL. Taken together our findings demonstrate the clinical utility of WGS as a diagnostic assay to inform and improve the management of adult B-Other ALL patients in the future.

Gamete Binning to Achieve Haplotype-Resolved Genome Assembly.

Haplotype-resolved genome assemblies remain a challenge in practice. Here, we provide a step-by-step guide on gamete binning, a method to generate haplotype-resolved genome assemblies for diploid species. The protocol starts by phasing heterozygous variants to individual haplotypes of specific chromosomes using the genome information of individual haploid gametes of the focal individual. Using phased variants, the whole-genome sequencing reads from the diploid genome can be genotyped and assigned into groups, which represent the individual haplotypes of each of the chromosomes. Finally, haplotype-specific chromosomes can be assembled independently using standard assembly tools. First applications of gamete binning revealed a haplotyping accuracy over 99%, which outperformed sequence-only or Hi-C-based haplotype-resolved genome assemblies.Availability: github.com/schneebergerlab/GameteBinning_prac .

Tandem duplication of a genomic region encoding glutathione S-transferase epsilon-2 and -4 genes in DDT-resistant Anopheles stephensi strain from India

The glutathione S-transferases (GST) genes are a multigene family of enzymes involved in the metabolism of endogenous and xenobiotic compounds by catalysing the conjugation of the reduced form of glutathione to the substrate. The epsilon class of GST (GSTe), unique to arthropods, is known to be involved in the detoxification process of several classes of insecticides, and GSTe2 in particular is known to have DDT dehydrochlorinase activity. This communication reports a tandem duplication of a genomic region encoding GSTe2 and GSTe4 genes in a laboratory-colonized DDT-resistant Anopheles stephensi. We identified duplication breakpoints and the organization of gene duplication through Sanger sequencing performed on long-PCR products. Manual annotation of sequences revealed a tandemly-arrayed duplication of a 3.62 kb segment of GST epsilon gene clusters comprised of five genes: a partial GSTe1, GSTe2, GSTe2-pseudogene, GSTe4 and partial GSTe5, interconnected by a conserved 2.42 kb DNA insert segment major part of which is homologous to a genomic region located on a different chromosome. The tandemly duplicated array contained a total of two GSTe2 and three GSTe4 functional paralog genes. Read-depth coverage and split-read analysis of Illumina-based whole-genome sequence reads confirmed the presence of duplication in the corresponding region of the genome. The increased gene dose in mosquitoes as a result of the GSTe gene-duplication may be an adaptive process to increase levels of detoxifying enzymes to counter insecticide pressure.

Mosaic Deletions of Known Genes Explain Skeletal Dysplasias With High and Low Bone Mass.

ABSTRACTMosaicism, a state in which an individual has two or more genetically distinct populations of cells in the body, can be difficult to detect because of either mild or atypical clinical presentation and limitations in the commonly used detection methods. Knowledge of the role of mosaicism is limited in many skeletal disorders, including osteopathia striata with cranial sclerosis (OSCS) and cleidocranial dysplasia (CCD). We used whole‐genome sequencing (WGS) with coverage >40× to identify the genetic causes of disease in two clinically diagnosed patients. In a female patient with OSCS, we identified a mosaic 7‐nucleotide frameshift deletion in exon 2 of AMER1, NM_152424.4:c.855_861del:p.(His285Glnfs*7), affecting 8.3% of the WGS reads. In a male patient with CCD, approximately 34% of the WGS reads harbored a 3710‐basepair mosaic deletion, NC_000006.11:g.45514471_45518181del, starting in intron 8 of RUNX2 and terminating in the 3′ untranslated region. Droplet digital polymerase chain reaction was used to validate these deletions and quantify the absolute level of mosaicism in each patient. Although constitutional variants in AMER1 and RUNX2 are a known cause of OSCS and CCD, respectively, the mosaic changes here reported have not been described previously. Our study indicates that mosaicism should be considered in unsolved cases of skeletal dysplasia and should be investigated with comprehensive and sensitive detection methods. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Hemophagocytic Lymphohistiocytosis Gene Variants in Childhood-Onset Systemic Lupus Erythematosus With Macrophage Activation Syndrome.

Macrophage activation syndrome (MAS), a life-threatening complication of systemic lupus erythematosus (SLE), resembles familial hemophagocytic lymphohistiocytosis (HLH), an inherited disorder of hyperinflammation. We compared the proportion of patients with childhood-onset SLE (cSLE) with and without MAS who carried low-frequency HLH nonsynonymous variants. We enrolled patients from the Lupus Clinic at SickKids, Toronto. Demographic and clinical features were extracted from the SLE database and ancestry was genetically inferred using multiethnic genotyping array data. Patients with MAS (based on expert diagnosis) underwent either paired-end whole-exome sequencing (WES; read depth: 70-118X) or whole-genome sequencing (WGS). Patients without MAS had WGS (read depth: 37-40X). In 16 HLH genes, we prioritized low-frequency (minor allele frequency [MAF] < 0.05) exonic nonsynonymous variants. We compared the proportion of patients with and without MAS carrying HLH variants (Fisher exact test, P < 0.05). MAFs were compared to an ancestrally matched general population (Trans-Omics for Precision Medicine [TOPMed] and Genome Aggregation Database [gnomAD]). The study included 81 patients with cSLE, 19 of whom had MAS. We identified 47 unique low-frequency nonsynonymous HLH variants. There was no difference in the proportion of patients with and without MAS carrying ≥ 1 HLH variants (37% vs 47%, P = 0.44). The MAS cohort did not carry more HLH variants when compared to an ancestrally matched general population. In a single-center multiethnic cSLE cohort, we found no difference in the proportion of patients with MAS carrying nonsynonymous HLH genetic variants compared to patients without MAS. To our knowledge, this is the first study to examine the frequency of HLH genetic variants in relation to MAS among patients with cSLE. Future studies are required to validate our findings.

Genomic and transcriptomic somatic alterations of hepatocellular carcinoma in non-cirrhotic livers

Genomic and transcriptomic somatic alterations of hepatocellular carcinoma in non-cirrhotic livers

In silico capsule locus typing for serovar prediction of Actinobacillus pleuropneumoniae.

Actinobacillus pleuropneumoniae is a causative agent of pleuropneumonia in pigs of all ages. A . pleuropneumoniae is divided into 19 serovars based on capsular polysaccharides (CPSs) and lipopolysaccharides. The serovars of isolates are commonly determined by serological tests and multiplex PCR. This study aimed to develop a genomic approach for in silico A. pleuropneumoniae typing by screening for the presence of the species-specific apxIV gene in whole-genome sequencing (WGS) reads and identifying capsule locus (KL) types in genome assemblies. A database of the A . pleuropneumoniae KL, including CPS synthesis and CPS export genes, was established and optimized for Kaptive. To test the developed genomic approach, WGS reads of 189 A . pleuropneumoniae isolates and those of 66 samples from 14 other bacterial species were analysed. ariba analysis showed that apxIV was detected in all 189 A . pleuropneumoniae samples. These apxIV-positive WGS reads were de novo assembled into genome assemblies and assessed. A total of 105 A . pleuropneumoniae genome assemblies that passed the quality assessment were analysed by Kaptive analysis against the A . pleuropneumoniae KL database. The results showed that 97 assemblies were classified and predicted as 13 serovars, which matched the serovar information obtained from the literature. The six genome assemblies from previously nontypable isolates were typed and predicted as serovars 17 and 18. Notably, one of the two “Actinobacillus porcitonsillarum” samples was apxIV positive, and its genome assembly was typed as KL03 with high identity and predicted as A . pleuropneumoniae serovar 3. Collectively, a genomic approach was established and could accurately determine the KL type of A . pleuropneumoniae isolates using WGS reads. This approach can be used with high-quality genome assemblies for predicting A . pleuropneumoniae serovars and for retrospective analysis.

K-mer based prediction of Clostridioides difficile relatedness and ribotypes.

Comparative analysis of Clostridioides difficile whole-genome sequencing (WGS) data enables fine scaled investigation of transmission and is increasingly becoming part of routine surveillance. However, these analyses are constrained by the computational requirements of the large volumes of data involved. By decomposing WGS reads or assemblies into k-mers and using the dimensionality reduction technique MinHash, it is possible to rapidly approximate genomic distances without alignment. Here we assessed the performance of MinHash, as implemented by sourmash, in predicting single nucleotide differences between genomes (SNPs) and C. difficile ribotypes (RTs). For a set of 1905 diverse C. difficile genomes (differing by 0–168 519 SNPs), using sourmash to screen for closely related genomes, at a sensitivity of 100 % for pairs ≤10 SNPs, sourmash reduced the number of pairs from 1 813 560 overall to 161 934, i.e. by 91 %, with a positive predictive value of 32 % to correctly identify pairs ≤10 SNPs (maximum SNP distance 4144). At a sensitivity of 95 %, pairs were reduced by 94 % to 108 266 and PPV increased to 45 % (maximum SNP distance 1009). Increasing the MinHash sketch size above 2000 produced minimal performance improvement. We also explored a MinHash similarity-based ribotype prediction method. Genomes with known ribotypes (n=3937) were split into a training set (2937) and test set (1000) randomly. The training set was used to construct a sourmash index against which genomes from the test set were compared. If the closest five genomes in the index had the same ribotype this was taken to predict the searched genome’s ribotype. Using our MinHash ribotype index, predicted ribotypes were correct in 780/1000 (78 %) genomes, incorrect in 20 (2 %), and indeterminant in 200 (20 %). Relaxing the classifier to 4/5 closest matches with the same RT improved the correct predictions to 87 %. Using MinHash it is possible to subsample C. difficile genome k-mer hashes and use them to approximate small genomic differences within minutes, significantly reducing the search space for further analysis.

Evaluating whole-genome sequencing quality metrics for enteric pathogen outbreaks.

BackgroundWhole genome sequencing (WGS) has gained increasing importance in responses to enteric bacterial outbreaks. Common analysis procedures for WGS, single nucleotide polymorphisms (SNPs) and genome assembly, are highly dependent upon WGS data quality.MethodsRaw, unprocessed WGS reads from Escherichia coli, Salmonella enterica, and Shigella sonnei outbreak clusters were characterized for four quality metrics: PHRED score, read length, library insert size, and ambiguous nucleotide composition. PHRED scores were strongly correlated with improved SNPs analysis results in E. coli and S. enterica clusters.ResultsAssembly quality showed only moderate correlations with PHRED scores and library insert size, and then only for Salmonella. To improve SNP analyses and assemblies, we compared seven read-healing pipelines to improve these four quality metrics and to see how well they improved SNP analysis and genome assembly. The most effective read healing pipelines for SNPs analysis incorporated quality-based trimming, fixed-width trimming, or both. The Lyve-SET SNPs pipeline showed a more marked improvement than the CFSAN SNP Pipeline, but the latter performed better on raw, unhealed reads. For genome assembly, SPAdes enabled significant improvements in healed E. coli reads only, while Skesa yielded no significant improvements on healed reads.ConclusionsPHRED scores will continue to be a crucial quality metric albeit not of equal impact across all types of analyses for all enteric bacteria. While trimming-based read healing performed well for SNPs analyses, different read healing approaches are likely needed for genome assembly or other, emerging WGS analysis methodologies.