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Minist?rio da Sa?de. Secretaria de Vigil?ncia em Sa?de. Instituto Evandro Chagas. Ananindeua, PA, Brasil. Minist?rio da Sa?de. Secretaria de Vigil?ncia em Sa?de. Instituto Evandro Chagas. Ananindeua, PA, Brasil. Minist?rio da Sa?de. Secretaria de Vigil?ncia em Sa?de. Instituto Evandro Chagas. Ananindeua, PA, Brasil. The resistance of hepatitis C virus (HCV) to direct-acting antiviral agents, used in chronic hepatitis C treatment, consists of a natural process resulting from resistance-associated substitutions (RASs) at specific amino acid regions. To identify and establish the natural prevalence of RASs in the NS3 gene in patients with chronic hepatitis C in the state of Par?, northern Brazil. Molecular analysis was performed on a total of 35 patients infected with HCV genotype 1, who were treatment-naive to protease inhibitors. HCV RNA was extracted from plasma and the NS3 region was amplified and submitted to DNA sequencing (Sanger). The general natural prevalence of RASs in the NS3 gene was 37.5 % (Y56F and S122T). The substitutions Y56F (34.3 %), S122T (3.1 %), V132I (15.6 %) and V170I (9.3 %) were identified. Y56F and S122T provide resistance to the protease inhibitors grazoprevir and simeprevir, respectively. All amino acid substitutions in the NS3 gene, including RASs, identified in patients from the state of Par? were present in other Brazilian studies. The natural presence of RASs in this study reflects the elevated genetic variability of HCV.

HIV-1 is characterized by its ability to mutate and recombine even at polymerase (pol) gene. However, pol-gene diversity is limited due to functional constraints. The aim of this study was to characterize longitudinally, by next-generation sequencing (NGS), HIV-1 variants based on pol-gene sequences, at intra- and inter-host level, from acute/early to chronic stages of infection, in the absence of antiretroviral therapy. Ten men who have sex with men (MSM) were recruited during primary infection and yearly followed for five years. Even after a maximum of a five-year follow-up period, the phylogenetic analysis of HIV-1 pol-gene sequences showed a host-defined structured pattern, with a predominance of purifying selection forces during the follow-up. MSM had been acutely infected by different HIV-1 variants mainly ascribed to pure subtype B, or BF recombinant variants and showed different genetic mosaicism patterns that last until the chronic stage, representing a major challenge for prevention strategies., Genetics; Microbiology; Virology; Viruses; Viral disease; Viral genetics; HIV-1; Phylogeny; Pol-gene; Next-generation-sequencing; Viral diversity; MSM.

We performed shotgun genome sequencing on a total of 19 different Musa genotypes including representatives of wild banana species Musa acuminata and M. balibisiana, allopolyploid bananas and plantains, Fe'i banana, pink banana (also known as hairy banana) and abacá (also known as hemp banana). We aligned sequence reads against a previously sequenced reference genome and assessed ploidy and, in the case of allopolyploids, the contributions of the A and B genomes; this provides important quality-assurance data about the taxonomic identities of the sequenced plant material. These data will be useful for phylogenetics, crop improvement, studies of the complex story of intergenomic recombination in AAB and ABB allotriploid bananas and plantains and can be integrated into resources such as the Banana Genome Hub.

Sex-specific markers are extremely important for unisexual breeding and understanding of sex determination mechanisms. Spotted mandarin fish (Siniperca scherzeri) and big eye mandarin fish (Siniperca kneri) are economically important freshwater fish in China. Here, for the first time, we successfully identified three male-specific markers in the two species. Through genome sequencing and bioinformatic analysis of three males, three females, one male mixed pool and one female mixed pool of spotted mandarin fish, we screened 27 potential male-specific sequences. After PCR validation, three male-specific markers were further identified. Moreover, these markers could also be used to identify genetic sex of spotted mandarin fish from different populations with 100 % accuracy. In addition, these markers were also successfully used in the identification of genetic sex of big eye mandarin fish. Notably, the sequences of male-specific markers were highly similar in the two species and the corresponding sequence of one marker was highly similar to partial genome sequence of amhy gene in mandarin fish (Siniperca chuatsi). In all, the verified male-specific markers suggested XY sex determination system existed in spotted mandarin fish and big eye mandarin fish and would play an important role in future work of sex control breeding.

Since the discovery of the ETV6-NTRK3 gene fusion in infantile fibrosarcoma two decades ago, it has become an important diagnostic marker because it is found in the majority of cases. However, the development of new molecular tests, including next generation sequencing, has uncovered additional gene fusions and other oncogenic mutations in tumors with the clinical and morphologic features of infantile fibrosarcoma. We present a case of infantile fibrosarcoma harboring a novel BMPR1A-RAF1 fusion and having a favorable outcome 6 years after surgery. This new structural alteration adds to the list of genetic aberrations in infantile fibrosarcoma and provides another example of the challenge of reconciling classic morphology with novel molecular genetics.

Food and waterborne protozoan pathogens can cause serious disease in people. Three common species Cryptosporidium parvum, Giardia enterica and Toxoplasma gondii can contaminate diverse shellfish species, including commercial oysters. Current methods of protozoan detection in shellfish are not standardized, and few are able to simultaneously identify multiple species. Here, we present a novel metabarcoding assay targeting the 18S rRNA gene followed by next generation sequencing (NGS) for simultaneous detection of Cryptosporidium spp., Giardia spp. and T. gondii spiked into oyster samples. We further developed a bioinformatic pipeline to process and analyze 18S rRNA data for protozoa classification. The ability of the NGS assay to detect protozoa was later compared with conventional PCR. Results demonstrated that background amplification of oyster and other eukaryotic DNA competed with that of protozoa for obtained sequence reads. Sequences of target protozoans were obtained across all spiking levels; however, low numbers of target sequences in negative controls imply that a threshold for true positives must be defined for assay interpretation. While this study focused on three target parasites, the ability of this approach to detect numerous known and potentially unknown protozoan pathogens make it a promising screening tool for monitoring protozoan contamination in food and water.

In 2019, 10 million new cases of tuberculosis have been reported worldwide. Our data reports genetic analyses of a Mycobacterium tuberculosis strain SBH321 isolated from a 31-year-old female with pulmonary tuberculosis. The genomic DNA of the strain was extracted from pure culture and subjected to sequencing using Illumina platform. M. tuberculosis strain SBH321 consists of 4,374,895 bp with G+C content of 65.59%. The comparative analysis by SNP-based phylogenetic analysis using maximum-likelihood method showed that our strain belonging to sublineage of the Ural family of Europe-America-Africa lineage (Lineage 4) and clustered with M. tuberculosis strain OFXR-4 from Taiwan. The whole genome sequence is deposited at DDBJ/ENA/GenBank under the accession WCJH00000000 (SRR10230353).

Acholeplasma laidlawii is widespread hypermutable bacteria (class Mollicutes) capable of infecting humans, animals, plants, which is the main contaminant of cell cultures and vaccine preparations. The mechanisms of the development of antimicrobial resistance of this bacterium are associated with the secretion of extracellular vesicles, which can mediate the lateral transfer of antibiotic resistance determinants. We compared the genome profiles of ciprofloxacin-resistant A.laidlawii strains PG8r1 (MIC 10 µg/ml) and PG8r3 (MIC 10 µg/ml) selected under different in vitro conditions - when ciprofloxacin-sensitive (MIC 0.5 µg/ml) A.laidlawii PG8B strain was cultured at increasing concentrations of ciprofloxacin in a broth medium alone, and with vesicles derived from the ciprofloxacin-resistant (MIC 20 µg/ml) A.laidlawii PG8R10c-2 strain, respectively. Genome profiles of PG8c-3 (obtained from a single colony of the strain PG8B) and PG8R10c-2 were analyzed too. Patterns of the quinolone target genes (gyrA, gyrB, parE, parC) containing in extracellular vesicles of PG8c-3, PG8R10c-2, PG8r1 and PG8r3 were determined. Genome sequencing was performed on the NextSeq Illumina platform. Search and annotation of single nucleotide polymorphisms were performed using Samtools and SnpEff, respectively. We also compared cellular proteomes of PG8c-3, PG8r1 and PG8r3. The cellular proteome profiles of the A. laidlawii strains were determined by two-dimensional gel electrophoresis and MALDI-TOF/TOF MS. This work presents data on single nucleotide polymorphisms (SNPs) found in the genomes of the ciprofloxacin-resistant strains selected under different in vitro conditions and proteins that were differentially expressed in the cells of ciprofloxacin-resistant strains selected under different conditions in vitro.

Background Whole exome sequencing and RNA sequencing (WES/RNASeq) should now be implemented in the clinical practice in order to increase access to optimal care for cancer patients. Providing results to Tumour Boards in a relevant time frame—that is, compatible with the clinical pathway—is crucial. Assessing the feasibility of this implementation in the French care system is the primary objective of the Multipli study, as one of the four pilot projects of the national France Genomic Medicine 2025 (FGM 2025) plan. The Multipli study encompasses two innovative trials which will be driven in around 2400 patients suffering from a soft-tissue sarcoma (Multisarc) or a metastatic colorectal carcinoma (Acompli). Methods Prior to launching the FGM 2025 cancer pilot study itself, the performance of the Multipli genomic workflow has been evaluated through each step, from the samples collection to the Molecular Tumour Board (MTB) report. Two Multipli-assigned INCa-labelled molecular genetics centres, the CEA-CNRGH sequencing platform and the Institut Bergonie’s Bioinformatics Platform were involved in a multicentric study. The duration of each step of the genomic workflow was monitored and bottlenecks were identified. Results Thirty barriers which could affect the quality of the samples, sequencing results and the duration of each step of the genomic pathway were identified and mastered. The global turnaround time from the sample reception to the MTB report was of 44 calendar days. Conclusion Our results demonstrate the feasibility of tumour genomic analysis by WES/RNASeq within a time frame compatible with the current cancer patient care. Lessons learnt from the Multipli WES/RNASeq Platforms Workflow Study will constitute guidelines for the forthcoming Multipli study and more broadly for the future clinical routine practice in the first two France Genomic Medicine 2025 platforms.

Objective Precision oncology depends on translating molecular data into therapy recommendations. However, with the growing complexity of next-generation sequencing-based tests, clinical interpretation of somatic genomic mutations has evolved into a formidable task. Here, we compared the performance of three commercial clinical decision support tools, that is, NAVIFY Mutation Profiler (NAVIFY; Roche), QIAGEN Clinical Insight (QCI) Interpret (QIAGEN) and CureMatch Bionov (CureMatch). Methods In order to obtain the current status of the respective tumour genome, we analysed cell-free DNA from patients with metastatic breast, colorectal or non-small cell lung cancer. We evaluated somatic copy number alterations and in parallel applied a 77-gene panel (AVENIO ctDNA Expanded Panel). We then assessed the concordance of tier classification approaches between NAVIFY and QCI and compared the strategies to determine actionability among all three platforms. Finally, we quantified the alignment of treatment suggestions across all decision tools. Results Each platform varied in its mode of variant classification and strategy for identifying druggable targets and clinical trials, which resulted in major discrepancies. Even the frequency of concordant actionable events for tier I-A or tier I-B classifications was only 4.3%, 9.5% and 28.4% when comparing NAVIFY with QCI, NAVIFY with CureMatch and CureMatch with QCI, respectively, and the obtained treatment recommendations differed drastically. Conclusions Treatment decisions based on molecular markers appear at present to be arbitrary and dependent on the chosen strategy. As a consequence, tumours with identical molecular profiles would be differently treated, which challenges the promising concepts of genome-informed medicine.

Introduction: Pathogenic variants in SURF1, a nuclear-encoded gene encoding a mitochondrial chaperone involved in COX assembly, are one of the most common causes of Leigh syndrome (LS). Material-methods: Sixteen patients diagnosed to have SURF1-related LS between 2012 and 2020 were included in the study. Their clinical, biochemical and molecular findings were recorded. 10/16 patients were diagnosed using whole-exome sequencing (WES), 4/16 by Sanger sequencing of SURF1, 1/16 via targeted exome sequencing and 1/16 patient with whole-genome sequencing (WGS). The pathogenicity of SURF1 variants was evaluated by phylogenetic studies and modelling on the 3D structure of the SURF1 protein. Results: We identified 16 patients from 14 unrelated families who were either homozygous or compound heterozygous for SURF1 pathogenic variants. Nine different SURF1 variants were detected The c.769G > A was the most common variant with an allelic frequency of 42.8% (12/28), c.870dupT [(p.Lys291*); (8/28 28.5%)], c.169delG [(p.Glu57Lysfs*15), (2/24; 7.1%)], c.532 T > A [(p.Tyr178Asn); (2/28, 7.1%)], c.653_654delCT [(p.Pro218Argfs*29); (4/28, 14.2%)] c.595_597delGGA [(p.Gly199del); (1/28, 3.5%)], c.751 + 1G > A (2/28, 4.1%), c.356C > T [(p.Pro119Leu); (2/28, 3.5%)] were the other detected variants. Two pathogenic variants, C.595_597delGGA and c.356C > T, were detected for the first time. The c.769 G > A variant detected in 6 patients from 5 families was evaluated in terms of phenotype-genotype correlation. There was no definite genotype – phenotype correlation. Conclusions: To date, more than 120 patients of LS with SURF1 pathogenic variants have been reported. We shared the clinical, molecular data and natural course of 16 new SURF1 defect patients from our country. This study is the first comprehensive research from Turkey that provides information about disease-causing variants in the SURF1 gene. The identification of common variants and phenotype of the SURF1 gene is important for understanding SURF1 related LS. Synopsis: SURF1 gene defects are one of the most important causes of LS; patients have a homogeneous clinical and biochemical phenotype. © 2020 The Authors, G0800674 Biotechnology and Biological Sciences Research Council, BBSRC: L016354 European Molecular Biology Organization, EMBO EMBO Türkiye Bilimsel ve Teknolojik Araştirma Kurumu, TÜBITAK Bilim Akademisi Newcastle upon Tyne Hospitals NHS Foundation Trust NIHR Bristol Biomedical Research Centre Lily Mae Foundation Medical Research Council, MRC, RM and RWT are supported by the Wellcome Centre for Mitochondrial Research ( 203105/Z/16/Z ), the Medical Research Council (MRC) International Centre for Genomic Medicine in Neuromuscular Disease, the Mitochondrial Disease Patient Cohort (UK) ( G0800674 ), Newcastle University Centre for Ageing and Vitality (supported by the Biotechnology and Biological Sciences Research Council and Medical Research Council L016354 ), UK NIHR Biomedical Research Centre for Ageing and Age-related disease award to the Newcastle upon Tyne Hospitals NHS Foundation Trust , the Lily Foundation and the UK NHS Specialist Commissioners which funds the “Rare Mitochondrial Disorders of Adults and Children” Diagnostic Service in Newcastle upon Tyne ( http://www.newcastle-mitochondria.com . OA is supported by the European Molecular Biology Organization (EMBO) Installation grant funded by the Scientific and Technological Research Council of Turkey (TUBITAK); Science Academy, Turkey; TUBITAK International Fellowship for Outstanding Researchers Programme.

This study compared the DNA sequences of cytochrome coxidase subunit I (COI) and histone H3 of Ephemeroptera, Odonata, Plecoptera, and Trichoptera in a pairwise manner, and calculated the sequence similarities based on uncorrected P-distance (number of identical sites in both sequences per total number of the sites compared). Datasets of annotated sequences, the source organisms of which are identified at the species level in taxonomy, were retrieved from INSD (GenBank/ ENA/ DDBJ) as of the end of May 2020. Similarity scores of the pairwise comparison were sorted by the combinations of taxonomic groups; intraspecific variations, intrageneric-interspecific divergences, intrafamily-intergeneric divergences, and intraorder-interfamily divergences for Ephemeroptera, Odonata, Plecoptera, and Trichoptera. Similarity scores at the cumulative relative frequency points (1%, 5%, 10%, and median) may be used as the threshold to differentiate between the taxonomic groups based on sequence match. This is often done in the characterization of morphologically-unidentified specimens using barcode sequences, in the metabarcoding analysis of the local fauna, and environmental DNA analysis.

Vibrio cholerae is a cause of serious endemic diarrhoea associated with cholera in many regions in the world. A total of 256 stool and rectal swabs were collected from patients suspected to have cholera admitted to three hospitals in Hillah, Babylon Governorate, Iraq, for the period 1 September to 29 December 2017. After the routine culture of samples for isolation and identification of V. cholerae isolates, PCR was performed for molecular detection of V. cholerae isolates based on 16S ribosomal RNA gene. Toxigenicity was detected by RTX toxin genes. PCR technique emphasized molecular detection of V. cholerae for eight isolates. Only two isolates (25%) possessed both the rtxA and rtxC genes, while only three isolates (37.5%) possessed the rtxB gene. DNA sequencing was performed for the eight isolates via analysis and phylogenetic tree. The observed bacterial variants were compared to their neighbour homologous reference sequences using the National Center for Biotechnology Information (NCBI) BLAST server (Basic Local Alignment Search Tool; https://blast.ncbi.nlm.nih.gov/Blast.cgi ). The findings indicated that the eight investigated isolates of V. cholerae were positioned in three different phylogenetic positions. Partial sequence dissimilarities were reported between GenBank isolate accession number MK212155.1 and these six clustered GenBank accession numbers of the same species. For the first time in Babylon Governorate, Iraq, the molecular assay, sequencing and phylogenetic tree are reported for V. cholerae and their toxins isolated during the 2017 cholera outbreak.

High throughput sequencing data collected from acid rock drainage (ARD) communities can reveal the active taxonomic and functional diversity of these extreme environments, which can be exploited for bioremediation, pharmaceutical, and industrial applications. Here, we report a seasonal comparison of a microbiome and transcriptome in Ely Brook (EB-90M), a confluence of clean water and upstream tributaries that drains the Ely Copper Mine Superfund site in Vershire, VT, USA. Nucleic acids were extracted from EB-90M water and sediment followed by shotgun sequencing using the Illumina NextSeq platform. Approximately 575,933 contigs with a total length of 1.54 Gbp were generated. Contigs of at least a size of 3264 (N50) or greater represented 50% of the sequences and the longest contig was 488,568 bp in length. Using Centrifuge against the NCBI “nt” database 141 phyla, including candidate phyla, were detected. Roughly 380,000 contigs were assembled and ∼1,000,000 DNA and ∼550,000 cDNA sequences were identified and functionally annotated using the Prokka pipeline. Most expressed KEGG-annotated microbial genes were involved in amino acid metabolism and several KEGG pathways were differentially expressed between seasons. Biosynthetic gene clusters involved in secondary metabolism as well as metal- and antibiotic-resistance genes were annotated, some of which were differentially expressed, colocalized, and coexpressed. These data can be used to show how ecological stimuli, such as seasonal variations and metal concentrations, affect the ARD microbiome and select taxa to produce novel natural products. The data reported herein is supporting information for the research article “Characterization of an acid rock drainage microbiome and transcriptome at the Ely Copper Mine Superfund site” by Giddings et al. [1] .

Breast cancer is the most common cancer in women, accounting for approximately 25% of all cancer cases worldwide. Some breast cancer patients are genetically predisposed to genes involved in genomic stability. We report the targeted genome sequencing data of 24 young women (aged below 45 years) breast cancer patients admitted to Cipto Mangunkusumo National Hospital, Jakarta, Indonesia. These data will be useful in detecting the genome markers of breast cancer and in deciding the diagnostics and therapies. DNA sequences were obtained using the Illumina NextSeq 500 platform. FASTQ raw files are available under BioProject accession number PRJNA606794 and Sequence Read Archive accession numbers SRR11774092–SRR11774115.

The complete genome of the thraustochytrid Aurantiochytrium limacinum strain CCAP_4062/1 was sequenced using both Illumina Novaseq 6000 and third generation sequencing technology PacBio RSII in order to obtain trustworthy assembly and annotation. The reads from both platforms were combined at multiple levels in order to obtain a reliable assembly, then compared to the A. limacinum ATCCⓇ MYA1381™ reference genome. The final assembly was annotated with the help of strain CCAP_4062/1 RNAseq data. A. limacinum strain CCAP_4062/1 is an industrial strain used for the production of very long chain polyunsaturated fatty acids, like the docosahexaenoic acid that is an essential fatty acid synthesised only at very low pace in humans and vertebrates . Thraustochytrids in general and Aurantiochytrium more specifically, are used for carotenoid and squalene production as well. Beside their biotechnological interest, thraustochytrids play a crucial role in both inshore and oceanic basins ecosystems. Genome sequences will foster biotechnological as well as ecological studies.

Freshwater catfish of the genus Clarias, known as the airbreathing catfish, are widespread and important for food security through small scale inland fisheries and aquaculture. Limited genomic data are available for this important group of fishes. The bighead catfish (Clarias macrocephalus) is a commercial aquaculture species in southeast Asia used for aquaculture and threatened in its natural environment through habitat destruction, over-exploitation and competition from other introduced species of Clarias. Despite its commercial importance and threats to natural populations, public databases do not include any genomic data for C. macrocephalus. We present the first genomic data for the bighead catfish from Illumina sequencing. A total of 128 Gb of sequence data in paired-end 150 bp reads were assembled de novo, generating a final assembly of 883 Mbp contained in 27,833 scaffolds (N50 length: 80.8 kbp) with BUSCO completeness assessments of 96.3% and 87.6% based on metazoan and Actinopterygii ortholog datasets, respectively. Annotation of the genome predicted 21,124 gene sequences, which were assigned putative functions based on homology to existing protein sequences in public databases. Raw fastq reads and the final version of the genome assembly have been deposited in the NCBI (BioProject: PRJNA604477, WGS: JAAGKR000000000, SRA: SRR11188453). The complete C. macrocephalus mitochondrial genome was also recovered from the same sequence read dataset and is available on NCBI (accession: MT109097), representing the first mitogenome for this species. Lastly, we find an expansion of the mb and ora1 genes thought to be associated with adaptations to air-breathing and a semi-terrestrial life style in this genus of catfish.

A study of 91 isolates from fish farms in Turkey showed that isolates P7T, P11, P24b, P29, P72, P73 and P158 belonged to the genus Pseudomonas according to 16S rRNA nucleotide sequence analysis. The analysis of the sequences of the RNA polymerase sigma factor gene (rpoD) located these strains in the Pseudomonas fluorescens lineage of species within the P. fluorescens subgroup, close to the cluster composed of the species Pseudomonas grimontii, Pseudomonas marginalis and Pseudomonas panacis. Based on similarities in the 16S rRNA and rpoD gene sequences of three previously isolated strains from other origins (CCUG 57209, CCUG 62357 and W5.2-93) linked them to the same cluster. A polyphasic taxonomic approach including phenotypic characterization, fatty acid composition, and multilocus sequence analysis, together with whole-cell MALDI-TOF data, corroborated this assumption. The genome G+C mol% contents were 59.48 and 59.71, respectively. The average nucleotide indices based on BLAST analysis and the genome-to-genome distance calculation for the P7T and CCUG 57209 strains with their closest relative, P. grimontii, were 88.16–88.29% and 38.10–38.20%, respectively. These data confirm that isolates P7T, P11, P24b, P29, P72, P73, P158, CCUG 57209, CCUG 62357 and W5.2-93 represent a new species for which the name Pseudomonas sivasensis is proposed, with P7T as a type strain (=CCUG 74260T= and CECT30107T)., This research was supported by the Scientific and Technological Research Council of Turkey (TUBITAK) [No: 118O420]. We are thankful to Bursa Uludag University for additional support.

Summary Decades of artificial selection have significantly improved performance and efficiency of animal production systems. However, little is known about the microevolution of genomes due to intensive breeding. Using whole-genome sequencing, we document dynamic changes of chicken genomes under divergent selection on adiposity over 19 generations. Directional selection reduced within-line but increased between-line genomic differences. We observed that artificial selection tended to result in recruitment of preexisting variations of genes related to adipose tissue growth. In addition, novel mutations contributed to divergence of phenotypes under selection but contributed significantly less than preexisting genomic variants. Integration of 15 generations genome sequencing, genome-wide association study, and multi-omics data further identified that genes involved in signaling pathways important to adipogenesis, such as autophagy and lysosome (URI1, MBL2), neural system (CHAT), and endocrine (PCSK1) pathways, were under strong selection. Our study provides insights into the microevolutionary dynamics of domestic animal genomes under artificial selection., Graphical Abstract, Highlights • Directional selection reduces within-line but increases between-line genomic difference • Artificial selection tends to recruit preexisting variations of genes for fatness • Novel mutations contribute to the divergence of fatness under artificial selection • Genes involved in signaling pathways important to adipogenesis are under selection, Biological Sciences; Evolutionary Biology; Genetics; Genomics

A Gram-negative bacterium, Pantoea stewartii subspecies stewartii (P. stewartii subsp. stewartii) has been recognized as the causative agent for jackfruit bronzing disease in Malaysia. Here, we report the whole genome sequencing dataset of P. stewartii subsp. stewartii strain SQT1 isolated from local infected jackfruit. The paired-end libraries with an insert size of 350 bp was subjected to the Illumina Hiseq 4000, generating a genome size of 4,783,993 bp with a G+C content of 53.7%. A total protein of 4,671 was identified including virulence factors, resistance factors and secretion systems. Pantoea stewartii subsp. stewartii strain DC283 (NCBI accession no. CP017581.1) was used as a reference genome, where the query hit 72% coverage and average sequencing depth of 68. In total, 28,717 nucleotide polymorphisms, 520 small insertion/deletions and 142 structure variants were identified. The complete genome was deposited at the European Nucleotide Archive under the sample accession number ERP119356 and study accession number PRJEB36196.

This dataset represents the root endophytic microbial community profile of maize (Zea mays L.), one of the largest food crops in South Africa, using a shotgun metagenomic approach. To the best of our understanding, this is the first account showcasing the endophytic microbial diversity in maize plants via the shotgun metagenomics approach. High throughput sequencing of the whole DNA from the community was carried out using NovaSeq 6000 system (Illumina). The data obtained consists of 10,915,268 sequences accounting for 261,906,948 bps with an average length of 153 base pairs and 43% Guanine+Cytosine content. The metagenome data can be accessed at the National Centre for Biotechnology Information SRA registered with the accession number PRJNA607664. Community analysis was done using an online server called MG-RAST, which showed that 0.12% of the sequences were archaeal associated, eukaryotes were 15.06%, while 84.77% were classified as bacteria. A sum of 28 bacterial, 22 eukaryotic and 4 archaeal phyla were identified. The predominant genera were Bacillus (16%), Chitinophaga (12%), Flavobacterium (4%), Chryseobacterium (4%), Paenibacillus (4%), Pedobacter (3%) and Alphaproteobacteria (3%). Annotation using Cluster of Orthologous Group (COG) revealed that 41.47% of the sequenced data were for metabolic function, 24.10% for chemical process and signaling, while 17.43% of the sequences were in the poorly characterized group. Annotation using the subsystem method showed that 18% of the sequences were associated with carbohydrates, 9% were for clustering-based subsystems, and 9% contain genes coding for amino acids and derivatives, which might be beneficial in plant growth and health improvement.

This dataset presents shotgun metagenomic sequencing of sunflower rhizosphere microbiome in Bloemhof, South Africa. Data were collected to decipher the structure and function in the sunflower microbial community. Illumina HiSeq platform using next generation sequencing of the DNA was carried out. The metagenome comprised 8,991,566 sequences totaling 1,607,022,279 bp size and 66% GC content. The metagenome was deposited into the NCBI database and can be accessed with the SRA accession number SRR10418054. An online metagenome server (MG RAST) using the subsystem database revealed bacteria had the highest taxonomical representation with 98.47%, eukaryote at 1.23%, and archaea at 0.20%. The most abundant genera were the Conexibacter (17%), Nocardioides (8%), Streptomyces (7%), Geodermatophilus (6%), Methylobacterium (5%), and Burkholderia (4%). MG-RAST assisted analysis also revealed functional annotation based on subsystem, carbohydrates sequence had 13.74%, clustering based subsystem 12.93%, amino acids and derivatives 10.30% coupled with other useful functional traits needed for plant growth and health.

Strain Marseille-Q0835T is an aerobic, non-motile and non-spore-forming Gram-positive coccus isolated from the stools of a Burkinabe woman. In this report, we present its phenotypic description including MALDI-TOF mass spectrometry analysis and genome sequencing. Strain Marseille-Q0835T; 2.9768-Mb genome exhibited a 41.9 mol% G+C content and 2699 predicted genes. Considering phenotypic features and comparative genome studies, we propose the strain Marseille-Q0835T as the type strain of Enterococcus burkinafasonensis sp. nov., a new species within the family Enterococcaceae.

Summary Kingdonia uniflora, an alpine herb, has an extremely narrow distribution and represents a model for studying evolutionary mechanisms of species that have adapted to undisturbed environments for evolutionarily long periods of time. We assembled a 1,004.7-Mb draft genome (encoding 43,301 genes) of K. uniflora and found significant overrepresentation in gene families associated with DNA repair, underrepresentation in gene families associated with stress response, and loss of most plastid ndh genes. During the evolutionary process, the overrepresentation of gene families involved in DNA repair could help asexual K. uniflora reduce the accumulation of deleterious mutations, while reducing genetic diversity, which is important in responding to environment fluctuations. The underrepresentation of gene families related to stress response and functional loss of ndh genes could be due to lack or loss of ability to respond to environmental changes caused by long-term adaptation to a relatively stable ecological environment., Graphical Abstract, Highlights • Provides a high-quality assembly for K. uniflora genome • K. uniflora shows overrepresentation in gene families associated with DNA repair • K. uniflora shows underrepresentation in gene families associated with stress response • The current endangered status of K. uniflora is related with adaptive degeneration, Biological Sciences; Plant Genetics; Plant Ecology; Plant Evolution

The data presented in this paper stand as supplementary information of the associated article “Karyological characterization of the common chameleon (Chamaeleo chamaeleon) provides insights on the evolution and diversification of sex chromosomes in Chamaeleonidae” [1] . This work provides (i) raw experimental data on the karyology of the common chameleon Chamaeleo chamaeleon and (ii) the results of bioinformatic analysis on sex-specific and repeated DNA sequences found in the same species. The karyological information here presented includes traditional staining method (Giemsa staining) and sequential C-banding + fluorochromes performed on Tunisian samples of the species. The sequence data include the alignments of the isolated DNA sequences with homologous sequences found in squamate Short Read Archives (SRAs) and the results of searches in public nucleic acid databases.

Vigna nakashimae is one of the closely related species of Vigna angularis (Adzuki bean). Two strain of ‘Ukushima’ and ‘G418’ were identified as salt tolerance strains in Vigna nakashimae from gene bank collection. F2 populations from an inter- or intra-specific cross between the sensitive and tolerant strains are useful for the detection of salt tolerance QTL in Vigna nakashimae. Although Vigna angularis reference genome is available and useful for genetic analysis by genotyping-by-sequencing/RADseq in closely related species, it is not enough for isolation of responsible genes. To reveal sequence variation in Vigna nakashimae “Ukushima” and “G418”, the whole genome sequencing was performed using Illumina HiSeq X Ten system (411,174,986 and 478,116,282 read). NGS data was deposited in the DNA Data Bank of Japan (DDBJ) under accession number DRA009307. Keywords: Vigna nakashimae, Closely wild relatives of Adzuki bean, Next-generation sequencing, HiSeq, Whole genome sequencing

Apple stem pitting virus (ASPV) mainly infects apple, pear and their closely related species in the world. ASPV causes some symptoms like leaf etiolation and stony pit in cultivated pear, but produces few symptoms in cultivated apple. We inoculated tobacco with ASPV, which originates from Nanking cherry (Prunus tomentosa), and we obtained tobacco RNA-sequencing data through high-throughput sequencing. In total, 17,401,736 clean reads were obtained after sequencing and removing adaptor sequences, contamination and low-quality reads. An RNA-seq data has been uploaded to Sequence Read Archive (SRA), which enables researchers to access the RNA-sequencing data of tobacco inoculated with ASPV. Keywords: Tobacco, Next-generation sequencing, RNA-seq, Apple stem pitting virus

Summary The diagnosis of Wilson disease is not always easy. For many patients, a combination of tests reflecting disturbed copper metabolism may be needed. Testing for ATP7B variants has become part of the routine diagnostic approach. The methods of genetic testing include analysis of the 21 coding exons and intronic flanking sequences, in which exons with recurrent variants would be prioritised depending on the mutation frequency in the local population. If sequencing the entire ATP7B gene cannot identify 2 variants and the suspicion for Wilson disease is high, after reviewing the clinical data, WES (whole-exome sequencing) or WGS (whole-genome sequencing) could be applied. A workflow based on the type and number of ATP7B variants responsible for Wilson disease is proposed. Genetic testing is indicated for confirmation of diagnosis, family screening, and screening of newborns and infants and in unclear cases suspected of suffering from Wilson disease. However, genetic testing is not a routine screening test for Wilson disease. If no additional variants can be identified, it can be assumed that other hereditary disorders may mimic Wilson disease (congenital disorders of glycosylation, MEDNIK syndrome, idiopathic or primary copper toxicoses).

Actinic keratosis (AK) arises on photo-damaged skin and is considered to be the precursor lesion of cutaneous squamous cell carcinoma (cSCC). Many findings support the involvement of β human papillomaviruses (HPVs) in cSCC, while very little is known on γ HPV types. The objective of this study was to characterize the spectrum of PV types in healthy skin (HS) and AK samples of the same immunocompetent individuals using next generation sequencing (NGS). Viral DNA of 244 AK and 242 HS specimens were amplified by PCR using two different sets of primers (FAP59/64 and FAPM1). Purified amplicons were pooled and sequenced using NGS. The study resulted in the identification of a large number of known β and γ PV types. In addition, 27 putative novel β and 16 γ and 4 unclassified PVs were isolated. HPV types of species γ-1 (e.g. HPV4) appeared to be strongly enriched in AK versus HS. The NGS analysis revealed that a large spectrum of known and novel PVs is present in HS and AK. The evidence that species γ-1 HPV types appears to be enriched in AK in comparison to HS warrants further studies to evaluate their role in development of skin (pre)cancerous lesions.

With advancements in the understanding of human cancers (carcinomas, sarcomas, and hematopoietic malignancies), molecular profiling, especially by Next Generation Sequencing (NGS), is playing an increasingly important role in the diagnosis, prognostication, and therapeutic management of cancer patients. The final and critical step in NGS is the annotation of detected variants and reporting of their clinical significance. Automated bioinformatics tools are available to assist with annotation, but the final responsibility for interpretation and validation of the annotation rests with the pathologist who may be constrained by the pressures of clinical sign-out and limited training in NGS. In this manuscript, we detail our experience in outsourcing variant annotation to a high-quality vendor to improve quality, standardize reporting, and decrease turn-around time of NGS reporting in clinical practice. We describe the composition of the evaluation team, steps that should be taken to evaluate potential annotation vendors, and detailed parameters that should be addressed before contracting with a vendor to guarantee the clinical reliability of the reported annotations.

Since the discovery of anaplastic lymphoma kinase fusion-positive (ALK+) NSCLC in 2007, the methods to detect ALK+ NSCLC have evolved and expanded from fluorescence in situ hybridization and immunohistochemistry to next-generation DNA sequencing, targeted RNA sequencing, and whole transcriptome sequencing. As such, the deep sequencing methods have resulted in the expansion of distinct fusion partners identified in ALK+ NSCLC to 90 (one variant PLEKHM2-ALK is found in small cell lung cancer but included in this catalog) by the end of January 2020; about 65 of them (since 2018) and most of the recent novel fusion partners were reported from China. Thirty-four of the distinct fusion partners are located on the short arm of chromosome 2; 28 of these 34 fusion partners are located on 2p21-25, in which ALK is located on 2p23.2-p23.1. Many of these new ALK+ NSCLC fusion variants have responded to ALK tyrosine kinase inhibitors (TKIs). Several of these novel ALK fusion variants were identified as being resistant to EGFR TKIs or as dual 3'ALK fusions. In addition, at least 28 intergenic ALK rearrangements have also been reported, with three of them reported as responding to crizotinib. This review aims to serve as a central source of reference of fusion partners in ALK+ NSCLC for clinicians and scientists. We aim to update and improve the list going forward.

Like any new technology, rapid integration of genomic sequencing (GS) into the clinical setting can pose challenges for genetic health professionals (GHPs) using it to diagnose patients. We conducted semi-structured interviews with 31 clinical geneticists and genetic counsellors across Europe, Australia and Canada to gain a better understanding of the issues they were experiencing when requesting GS and receiving reports from laboratories. There was a spectrum of interactions between genetic health professionals and laboratories. This ranged from those that almost exclusively request sequencing from the laboratory that is affiliated with their genetic service, to those who do not have access to exome sequencing 'in-house' and instead send patient samples to a selection of different external laboratories. In general, a closer interaction between the clinicians and the laboratory scientists increased the involvement of the clinicians in the analysis/interpretation process. This appeared to lead to fewer, but more clinically relevant variants being reported, and greater GHP satisfaction in what is reported. Our findings suggest that GHPs consider integration of clinical expertise into the analysis/interpretation process is critical to ensure that the variants reported are of high clinical significance to patients. They also highlight the importance of providing GHPs with training in report interpretation. ispartof: EUROPEAN JOURNAL OF MEDICAL GENETICS vol:63 issue:3 ispartof: location:Netherlands status: published

Cancer is a sickness, initiated and driven via accumulation and interaction of genetic and epigenetic modification of genes involved in regulation of cell communication, signaling and growth division. Epigenetics is known as heritable changes in the expression of genes which are different from mutations that are not attributable to modification of DNA sequence. There are two leading epigenetic mechanisms; histone modification and DNA methylation. The acts of epigenetics in controlling of gene expression have been considered as elementary process in the pathogenesis of a variety of malignancies, for instance reproductive organs cancers. Recently, numerous exciting discoveries in epigenetics field have been focused in the study of reproductive cancers organs in females. Epigenetic modifications in the DNA of cancer patients and precancerous lesions provide the promise of novel biomarkers for early cancer prediction, diagnosis, prognosis, and treatment response. In addition, various epigenetic modifications represent potential targets of novel therapeutic strategies and treatments of cancer. In the future, pioneering diagnostic methodologies, technologies and treatment regimens will be based on epigenetic mechanisms and may be integrated into the gynecologist's practice. Keywords: Cancer, DNA methylation, Epigenetics, Histones modifications, Female, Urogenital organs

11 pages, 5 figures, 2 tables, supplementary data https://doi.org/10.1016/j.ecss.2019.106565, Organic nutrient loading in coastal waters has considerably increased due to anthropogenic activities over the last decades. In particular, the increase in the proportion of organic vs. inorganic nitrogen may have an impact on microbial plankton communities, particularly within the small size classes, where mixotrophy is likely to be widespread. Here we explore the change in taxonomic composition of small eukaryotes (, This research was supported by the Xunta de Galicia through DIMENSION project (grant EM2013/023) and by Spanish Ministry of Economy, Industry and Competitivity (MINECO) trough ENVISION project (grant CTM2014-59031-P) in collaboration with the “Instituto Español de Oceanografía” (IEO) RADIALES project. Esther Barber-Lluch was funded by an F.P.I. MINECO fellowship. [...] R. Logares was supported by a Ramón y Cajal fellowship (RYC-2013-12554, MINECO, Spain), With the funding support of the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S), of the Spanish Research Agency (AEI)

Understanding multicellular physiology and pathobiology requires analysis of the relationship between genotype, chromatin organisation and phenotype. In the multi-omics era, many methods exist to investigate biological processes across the genome, transcriptome, epigenome, proteome and metabolome. Until recently, this was only possible for populations of cells or complex tissues, creating an averaging effect that may obscure direct correlations between multiple layers of data. Single-cell sequencing methods have removed this averaging effect, but computational integration after profiling distinct modalities separately may still not completely reflect underlying biology. Multiplexed assays resolving multiple modalities in the same cell are required to overcome these shortcomings and have the potential to deliver unprecedented understanding of biology and disease.

Blastocystis sp. is an intestinal protozoan commonly found in fecal samples of many animal species, including humans, but poorly studied in transplant candidates. The aim of this study was to evaluate the occurrence and molecular identification of Blastocystis sp. in fecal samples from transplant candidates. A polymerase chain reaction was performed using specific primers for Blastocystis ribosomal DNA. The DNA sequences obtained were aligned and compared with other sequences from the GenBank and MLST databases. The analyzed samples showed a positivity of 16% (24 of 150) for Blastocystis sp. The highest occurrence was observed in renal transplant candidates (31.4%), followed by hepatic transplant candidates (10.4%) and candidates for bone marrow transplantation (5.9%). Subtype (ST) 3 (45.8%) was the most prevalent among the isolates, followed by ST1 (37.5%), ST2 (12.5%), and ST7 (4.2%). This is the first study of molecular identification Blastocystis sp. in transplant candidates. Our results confirmed that ST3 was the most common subtype in transplant candidates and reinforce the importance of new studies to investigate of Blastocystis sp. in these patients. Keywords: Blastocystis sp., Subtype, Transplant candidate, Brazil

Summary The DNA of eukaryotes is wrapped around histone octamers to form nucleosomes. Although it is well established that the DNA sequence significantly influences nucleosome formation, its precise contribution has remained controversial, partially owing to the lack of quantitative affinity data. Here, we present a method to measure DNA-histone binding free energies at medium throughput and with high sensitivity. Competitive nucleosome formation is achieved through automation, and a modified epifluorescence microscope is used to rapidly and accurately measure the fractions of bound/unbound DNA based on fluorescence anisotropy. The procedure allows us to obtain full titration curves with high reproducibility. We applied this technique to measure the histone-DNA affinities for 47 DNA sequences and analyzed how the affinities correlate with relevant DNA sequence features. We found that the GC content has a significant impact on nucleosome-forming preferences, but 10 bp dinucleotide periodicities and the presence of poly(dA:dT) stretches do not., Graphical Abstract, Highlights • Robotics permits full titration series to measure histone-DNA binding affinities • Fluorescence anisotropy used as a fast, sensitive readout of bound/unbound DNA • Free energies span three orders of magnitude, less for naturally occurring sequences • GC content is a major determinant of measured binding free energies, Molecular Biology; Molecular Interaction; Biological Sciences Research Methodologies

Over the past few decades the development of new molecular technologies has revolutionized diagnosis in the reproductive medicine field, with the evaluation of the two main factors involved in a successful pregnancy: the embryo and the endometrium. The detection of genetically abnormal embryos, as well as the identification of an optimum endometrium using transcriptomics have become a priority in assisted reproductive treatments to increase pregnancy rates. This chapter provides an overview of the molecular techniques currently employed in assisted reproduction for embryo evaluation such as preimplantation genetic testing karyotyping, fluorescence in situ hybridization, polymerase chain reaction, comparative genomic hybridization array, single-nucleotide polymorphism array, karyomapping, and next-generation sequencing; and for endometrial evaluation through gene expression—real-time polymerase chain reaction, DNA microarrays, and RNA-Seq. In each section, the reader will find a detailed description of the molecular principles of each technique, together with examples of its applications in clinical practice.

The brain contains immense cellular diversity. Early neuroanatomists identified broad structural brain features and used a variety of stains to characterize, draw, and name a number of constituent cell types. However, our granular understanding of what defines cellular identity, and how this relates to human neurogenesis, has been transformed by the sequencing of the human genome and subsequent technological advances. The genome enabled a translation of single gene level studies to genome-wide, unbiased characterization of gene expression patterns. Rapidly developing gene sequencing technology has revolutionized our understanding of cellular diversity, first based on microarray technology, then RNA-sequencing of 3′ fragments, to more recent whole-transcriptome sequencing, and ultimately single-cell sequencing. Single-cell sequencing allows for a heterogeneous tissue, like the brain, to be broken down into its component cell types based on fundamentally relevant single cell level gene programs. The advent of single-cell sequencing has been transformative for many fields, but using single-cell sequencing in the study of the brain and specifically brain development has enabled a convergence of the understanding of gene expression with features such as area, layer, age, connectivity, morphology, and ultimately cellular function, to achieve a core foundational understanding of cell type identity. While a number of these approaches are now being applied to human development, they have already been widely applied to rodent models, especially the mouse. Due to limitations of accessibility of human brain tissue, in vitro model systems such as the cerebral organoid have been developed to further our understanding of cellular and molecular developmental programs and to functionally test hypotheses gathered from sequencing studies. Here, we focus on established and emerging single-cell technologies, how they have enabled discovery in human brain development, and how organoid models recapitulate cellular developmental trajectories.

Nowadays, there are strong efforts in developing new technology for rapid detection of specific DNA sequences for environmental monitoring, forensic analysis and rapid biomedical diagnosis applications. That is where microfluidic paper-based analytical devices are positioned as suitable platforms for the development of point of care analytical devices, due to their simple fabrication protocols, ease of use and low cost. Herein, a methodology for in situ single strand DNA detection by using a colorimetric assay based on the formation of a DNAzyme within a paper substrate was developed. A DNAzyme that could only be formed in the presence of a specific sequence of the Y human amelogenin gene was designed. The performance of the DNAzyme was followed colorimetrically first in solution and then in paper substrates. The reaction was found to be specific to the Y fragment selected as analyte. The DNAzyme reaction on paper enabled the unequivocal colorimetric identification of the Y single strand DNA fragment both qualitatively, with the naked eye (143 ng), and quantitatively by image analysis (45.7 ng). As a proof of concept, a microfluidic paper-based device, pre-loaded with all DNAzyme reagents, was characterized and implemented for the simultaneous detection of X and Y single strand DNA fragments. This work was supported by Gobierno de España, Ministerio de Economía y Competitividad, with Grant No. BIO2016-80417-P; funding from Basque Government (Grant: IT1271-19) and European Union funds: DNASURF (H2020-MSCA-RISE-778001). E.A.-H. acknowledges funding from the Basque Government, Department of Education, for predoctoral fellowship 2016.

Temperature stress has an adverse effect on crop productivity worldwide. With the advancements of genome sequencing, the complex stress-response mechanisms in plants are better understood, and important candidates have been identified. microRNAs (miRNAs) and endogenous small interfering RNAs (siRNAs) are the new regulators that have been identified to play a role in small RNA-based regulation of gene expression during stress responses. These findings have broadened our understanding of stress responses in plants, and important functional roles have been discovered for these small RNAs in cold stress response. Here, we summarize and highlight new findings vis-a-vis the roles of small RNAs in plant cold stress response and acclimation. This understanding of small RNA (miRNAs and endogenous siRNAs)-mediated cold stress tolerance can be extrapolated to other plant species, exploring the outlook for using these small RNAs in molecular breeding and genetic engineering programs for improvement of cold stress tolerance in plants.

The genome of any living organism is the foundation upon which every aspect of that organism’s life is built, including all physical characteristics and interactions with the environment, as well as the mechanisms by which the genome sequence content is passed to offspring. The citrus genome underlies and influences every aspect of citrus trees and fruit, and therefore nearly every chapter topic that follows in this book may best be understood when viewed through the lens of genomic comprehension. A brief history of citrus genome sequencing activities is presented in the historical context of rapidly evolving improvements in genome sequencing technologies, including a summary of de novo genome sequence assemblies. Examples of how the abundance of available citrus genome sequence information has informed studies on the nature of progenitor citrus species, and the contemporary citrus species and the admixtures represented in modern citrus cultivars. Further, the specifics of comparative genome analyses and the conclusions drawn on the evolution and domestication of mandarins are provided. Sequencing technology and associated informatics will continue to improve in the coming years, and the application of the new fundamental information to be generated will support and inform future advances in all the subject areas that follow in this book.

In this chapter, secondary findings are discussed in the context of exome and genome sequencing that is being conducted to identify genetic variation in individuals suspected to have genetic disease. We summarize how secondary findings are discovered in clinical large-scale DNA sequencing and the sequencing technologies that are currently being employed by clinical and research laboratories. Moreover, we detail the downstream analytical pipelines that are utilized to detect potentially medically relevant genetic variation and discuss places within this pipeline where secondary genetic findings can be proactively sought out or even avoided.

Methyl-DNA immunoprecipitation is used in combination with next generation sequencing (MeDIP-seq) as a genome-wide approach to enrich for and analyze methylated DNA. Fragmented DNA containing methylated cytosines is purified from genomic samples via an antibody to 5-methylcytosine (5mC), then sequenced and analyzed in order to detect peaks of differential methylation across the genomic landscape. Introduced more than a decade ago, MeDIP enrichment is considered a useful, repeatable, and affordable approach for methylation research. As next generation sequencing has become increasingly accessible and affordable, deep sequencing of the enriched DNA has replaced the initial use of microarrays. At this time, researchers consider MeDIP-seq a viable protocol to answer most questions about DNA methylation, providing single-base resolution is not required. Many well-established downstream analysis tools are also available, making MeDIP-seq a consistently popular option. As we continue to engage with highly complex methylomes, which may vary tremendously by cell-type, timepoint, and environment, we must focus on proper study design, larger cohorts, and judicious deployment of experimental controls in order to pull meaningful signals from background noise.

The application of sensitive genetic technologies, capable of detecting mutations and chromosome abnormalities in minute tissue samples (and even single cells), has provided an unprecedented insight into the molecular mechanisms governing the earliest stages of human development. In a clinical context, the utilisation of these methods has allowed detection of disease causing mutations and aneuploidies in preimplantation embryos generated using in vitro fertilisation (IVF) technology, assisting patients seeking to avoid the transmission of serious inherited conditions and also aiding infertile couples hoping to maximise the chances of achieving a viable pregnancy when having IVF treatment. Preimplantation genetic testing (PGT), as this approach is known, has undergone much development during the thirty years that it has been available and continues to evolve. There has been increasing interest in the analysis of embryonic DNA found in the medium in which the embryo is cultured or in the fluid that fills the blastocoel cavity at the blastocyst stage of development. The aim is to use this material to perform a genetic assessment of the embryo, thereby avoiding the need to biopsy any of its cells. In theory, this should significantly reduce the costs of PGT related to equipment and labour and eliminate any risk to the embryo associated with biopsy. Methods for non-invasive PGT (niPGT) are still in being optimised, but thus far reported accuracy rates have been inferior to those achieved using embryo biopsy. It is clear that further technical development is needed if non-invasive methods of genetic testing are to replace or supplement traditional forms of PGT. Another way to reduce the costs of PGT is to harness powerful new DNA sequencing technologies in order to create methods that allow the detection of multiple pathogenic mutations with a single protocol. Taken to its ultimate extent, this could involve sequencing of the entire genome of the embryo biopsy specimens, although this remains relatively expensive at the current time. While such an approach could deliver a comprehensive genetic evaluation of a patient’s embryos, there are technical challenges, not only with the generation of accurate sequence data, but also with the clinical interpretation of any variants detected. The possibility that the entire genome of embryos could be revealed, and that the information could be used to select one for transfer to the uterus over another, also raises ethical concerns. Controversially, PGT has recently been applied to polygenic disorders, where combinations of genetic factors each contribute to an overall risk of disease. In most cases, such tests are not diagnostic and instead look at the relative probability of developing disease, which in many cases have a late onset. While PGT for polygenic disorders and the prospect of whole genome sequencing of embryos have been the subjects of much debate, perhaps the most ethically challenging development in the field of preimplantation genetics concerns the potential clinical application of genome editing technologies. Such methods hold great promise as a means to correct inherited mutations associated with serious conditions and would allow the discard of affected embryos to be avoided. However, the safety of these methods remains to be determined and application at the preimplantation stage is especially controversial due to the high likelihood that the germinal cells would be altered, meaning that any changes would be transmitted to future generations. Genome editing has enormous research and clinical potential, but more work is needed to confirm its safety, efficacy and ethical desirability when applied to gametes and preimplantation embryos.