Comprehensive characterization of copy number variation (CNV) called from array, long- and short-read data

Background SNP arrays, short- and long-read genome sequencing are genome-wide high-throughput technologies that may be used to assay copy number variants (CNVs) in a personal genome. Each of these technologies comes with its own limitations and biases, many of which are well-known, but not all of them are thoroughly quantified. Results We assembled an ensemble of public datasets of published CNV calls and raw data for the well-studied Genome in a Bottle individual NA12878. This assembly represents a variety of methods and pipelines used for CNV calling from array, short- and long-read technologies. We then performed cross-technology comparisons regarding their ability to call CNVs. Different from other studies, we refrained from using the golden standard. Instead, we attempted to validate the CNV calls by the raw data of each technology. Conclusions Our study confirms that long-read platforms enable recalling CNVs in genomic regions inaccessible to arrays or short reads. We also found that the reproducibility of a CNV by different pipelines within each technology is strongly linked to other CNV evidence measures. Importantly, the three technologies show distinct public database frequency profiles, which differ depending on what technology the database was built on.

rmvPFBAM: Removing Primers from BAM Files Based on Amplicon-Based Next-Generation Sequencing and Cloud Computing When Analyzing Personal Genome Data

Personal genomic data constitute one important part of personal health data. However, due to the large amount of personal genomic data obtained by the next-generation sequencing technology, special tools are needed to analyze these data. In this article, we will explore a tool analyzing cloud-based large-scale genome sequencing data. Analyzing and identifying genomic variations from amplicon-based next-generation sequencing data are necessary for the clinical diagnosis and treatment of cancer patients. When processing the amplicon-based next-generation sequencing data, one essential step is removing primer sequences from the reads to avoid detecting false-positive mutations introduced by nonspecific primer binding and primer extension reactions. At present, the removing primer tools usually discard primer sequences from the FASTQ file instead of BAM file, but this method could cause some downstream analysis problems. Only one tool (BAMClipper) removes primer sequences from BAM files, but it only modified the CIGAR value of the BAM file, and false-positive mutations falling in the primer region could still be detected based on its processed BAM file. So, we developed one cutting primer tool (rmvPFBAM) removing primer sequences from the BAM file, and the mutations detected based on the processed BAM file by rmvPFBAM are highly credible. Besides that, rmvPFBAM runs faster than other tools, such as cutPrimers and BAMClipper.

Human Genetic Diversity Alters Therapeutic Gene Editing Off-Target Outcomes

CRISPR gene editing holds great promise to modify somatic genomes to ameliorate disease. In silico prediction of homologous sites coupled with biochemical evaluation of possible genomic off-targets may predict genotoxicity risk of individual gene editing reagents. However, standard computational and biochemical methods focus on reference genomes and do not consider the impact of genetic diversity on off-target potential. Here we developed a web application called CRISPRme that explicitly and efficiently integrates human genetic variant datasets with orthogonal genomic annotations to predict and prioritize off-target sites at scale. The method considers both single-nucleotide variants (SNVs) and indels, accounts for bona fide haplotypes, accepts spacer:protospacer mismatches and bulges, and is suitable for personal genome analyses. We tested the tool with a guide RNA (gRNA) targeting the BCL11A erythroid enhancer that has shown therapeutic promise in clinical trials for sickle cell disease (SCD) and β-thalassemia (Frangoul et al. NEJM 2021). We find that the top predicted off-target site is produced by a non-reference allele common in African-ancestry populations (rs114518452, minor allele frequency (MAF) = 4.5%) that introduces a protospacer adjacent motif (PAM) for SpCas9. We validate that SpCas9 generates indels (~9.6% frequency) and chr2 pericentric inversions in a strictly allele-specific manner in edited CD34+ hematopoietic stem/progenitor cells (HSPCs), although a high-fidelity Cas9 variant mitigates this off-target. This report illustrates how population and private genetic variants should be considered as modifiers of genome editing outcomes. We expect that variant-aware off-target assessment will be required for therapeutic genome editing efforts going forward, including both ongoing and future clinical trials, and we provide a powerful approach for comprehensive off-target prediction. CRISPRme is available at crisprme.di.univr.it. Disclosures No relevant conflicts of interest to declare.

1001. Chronic Colonization with Toxigenic Clostridioides difficile Strains Drives Colonic Tumorigenesis in Mice

Background Long-term effects of chronic and/or recurrent C. difficile infections (CDI) are not well understood, and any potential role of CDI in colorectal cancer (CRC) risk is presently unknown. While pursuing efforts to identify novel procarcinogenic microbes, we identified two mucosal slurries from CRC patients (3728T and 3752T) that were tumorigenic in germ-free (GF) ApcMin/+ mice. Surprisingly, both of these CRC patient slurries were positive for C. difficile by 16S rRNA amplicon sequencing. Given the ability of other chronic infections to promote tumorigenesis (e.g., H. pylori), we hypothesized that chronic colonization with C. difficile could promote tumorigenesis in the colon. Methods A consortium of 30 bacterial isolates including a toxigenic tcdA+ tcdB+ C. difficile strain (CIm_3728T) was cultured from GF ApcMin/+ mice gavaged with the 3728T slurry. This consortium was gavaged into additional GF ApcMin/+ mice with or without C. difficile strains CIm_3728T, CIm_3752T (isolated from mice gavaged with the 3752T slurry), or isogenic tcdA/tcdB mutants of the M7404 R027 strain. Single cell RNA sequencing (scRNAseq), high dimensional (HD) flow cytometry, and fluorescence in situ hybridization (FISH) with EUB338 and Cd198 probes were performed on distal colons from mice gavaged with either complex CRC slurries or the 3728T isolates with CIm_3728T. Results C. difficile strains drove tumorigenesis of the 3728T isolate mixture (Fig. 1A,B). Tumorigenesis was associated with early procarcinogenic signaling and spatial changes including induction of Wnt signaling in colonic epithelial progenitor cells by scRNAseq, IL-17 induction in immune cells by HD flow cytometry, and bacterial biofilm invasion deep into epithelial crypts by FISH. Tumorigenesis correlated with chronic colonization with toxigenic strains of C. difficile and was toxin-dependent, as toxin mutant strains (M7404 tcdA-tcdB-) did not induce tumors. Figure 1. C. difficile strains from CRC patients induce distal colonic tumorigenesis in germ-free (GF) ApcMin/+ mice. A consortium of 30 bacteria, including C. difficile, were isolated from mice gavaged with the 3728T human CRC mucosal slurry. These isolates were then gavaged into additional GF ApcMin/+ mice, with or without C. difficile isolates from mice gavaged with the 3728T slurry or 3752T slurry. (A) Colonic tumor numbers in GF ApcMin/+ mice at 10 wk p.i. demonstrate that C. difficile (Cd) drives the tumorigenesis of this 30-member bacterial consortium. (B) Gross tumors can be observed in the colon of a representative mouse gavaged with the 3728T isolates with the CIm_3728T (top) or CIm_3752T (middle) strain of C. difficile but not in a mouse gavaged with the isolates lacking C. difficile (bottom). Conclusion Toxigenic C. difficile strains isolated from human CRC mucosal slurries were pro-carcinogenic in mice, suggesting that C. difficile is a potential driver of CRC. Given the public health burden of C. difficile, further studies are warranted to determine whether C. difficile infections (initial, recurrent, and chronic asymptomatic) increase CRC risk in patients. Disclosures Jada Domingue, PhD, AstraZeneca (Employee) James White, PhD, Personal Genome Diagnostics (Consultant) Patricia J. Simner, PhD, Accelerate Diagnostics (Grant/Research Support)Affinity Biosensors (Grant/Research Support)BD Diagnostics (Consultant, Grant/Research Support)GeneCapture (Consultant)OpGen, Inc (Consultant, Grant/Research Support)Shionogi, Inc (Consultant) Karen C. Carroll, MD, MeMed (Scientific Research Study Investigator)Meridian Diagnostics, Inc. (Grant/Research Support)Pattern Diagnostics (Advisor or Review Panel member)Scanogen, Inc. (Advisor or Review Panel member) Karen C. Carroll, MD, Pattern Diagnostics, Inc. (Individual(s) Involved: Self): Grant/Research Support; Scanogen, Inc. (Individual(s) Involved: Self): Consultant Cynthia L. Sears, MD, Bristol Myers Squibb (Grant/Research Support)Ferring (Advisor or Review Panel member)Janssen (Grant/Research Support)

Molecular epidemiologic characteristics of hemagglutinin from five waves of avian influenza A (H7N9) virus infection, from 2013 to 2017, in Zhejiang Province, China

There have been five waves of influenza A (H7N9) epidemics in Zhejiang Province between 2013 and 2017. Although the epidemiological characteristics of the five waves have been reported, the molecular genetics aspects, including the phylogeny, evolution, and mutation of hemagglutinin (HA), have not been systematically investigated. A total of 154 H7N9 samples from Zhejiang Province were collected between 2013 and 2017 and sequenced using an Ion Torrent Personal Genome Machine. The starting dates of the waves were 16 March 2013, 1 July 2013, 1 July 2014, 1 July 2015, and 1 July 2016. Single-nucleotide polymorphisms (SNPs) and amino acid mutations were counted after the HA sequences were aligned. The evolution of H7N9 matched the temporal order of the five waves, among which wave 3 played an important role. The 55 SNPs and 14 amino acid mutations with high frequency identified among the five waves revealed the dynamic occurrence of mutation in the process of viral dissemination. Wave 3 contributed greatly to the subsequent epidemic of waves 4 and 5 of H7N9. Compared with wave 1, wave 5 was characterized by more mutations, including A143V and R148K, two mutations that have been reported to weaken the immune response. In addition, some amino acid mutations were observed in wave 5 that led to more lineages. It is necessary to strengthen the surveillance of subsequent H7N9 influenza outbreaks.

TT-Mars: Structural Variants Assessment Based on Haplotype-resolved Assemblies

Variant benchmarking is a critical component of method development and evaluating the accuracy of studies of genetic variation. Currently, the best approach to evaluate the accuracy of a callset is the comparison against a well curated gold standard. In repetitive regions of the genome it may be difficult to establish what is the truth for a call, for example when different alignment scoring metrics provide equally supported but different variant calls in on the same data. Here we provide an alternative approach, TT-Mars, that takes advantage of the recent production of high-quality haplotype-resolved genome assemblies by evaluating variant calls based on how well their call reflects the content of the assembly, rather than comparing calls themselves. We used TT-Mars to assess callsets from different SV discovery methods on multiple human genome samples and demonstrated that it is capable at accurately classifying true positive and false positive SVs. On the HG002 personal genome, TT-Mars recapitulates 96.0%-99.6% of the validations made using the Genome in a Bottle gold standard callset evaluated by truvari, and evaluates an additional 121-10,966 variants across different callsets. Furthermore, with a group of high-quality assemblies, TT-Mars can evaluate performance of SV calling algorithms as a distribution rather than a point estimate. We also compare TT-Mars against the long-read based validation tool, VaPoR, and when assembly-based variant calls produced by dipcall are used as a gold standard. Compared with VaPoR, TT-Mars analyzes more calls on a long read callset by assessing more short variant calls (< 100 bases), while requiring smaller input. Compared with validation using dipcall variants, TT-Mars analyzes 1,497-2,229 more calls on long read callsets and has favorable results when candidate calls are fragmented into multiple calls in alignments. TT-Mars is available at https://github.com/ChaissonLab/TT-Mars.git with accompanying assembly data and corresponding liftover files.

PGP1 personal genome assembly - a hybrid assembly dataset using ONT′s PromethION and PacBio′s HiFi sequencing

PGP1 is the first participant of Personal Genome Project. We present the PGP1′s chromosome-scale genome assembly. It was constructed using 255 Gb ultra-long PromethION reads and 97 Gb short paired-end reads. For reducing base calling errors, we corrected PromethION reads using 72 Gb PacBio HiFi reads. 327 Gb Hi-C chromosomal mapping data were utilized to maximize the assembly′s contiguity. PGP1′s contig assembly was 3.01 Gb in length comprising of 4,234 contigs with an N50 value of 33.8 Mb. After scaffolding with Hi-C data and extensive manual curation, we obtained a chromosome-scale assembly that represents 3,880 scaffolds with an N50 value of 142 Mb. From the Merqury assessment, PGP1 assembly achieved a high QV score of Q45.45. For a gene annotation, we predicted 106,789 genes with a liftover from the Gencode 38 and an assembly of transcriptome data.

A virtual deliberative public engagement study on heritable genome editing among South Africans: Study protocol

This article outlines the protocol for a prospective study for virtual deliberative public engagement on heritable genome editing in humans. The study intends to create a platform for a diverse group of 25–30 South Africans to engage with a facilitator and each other on 15 policy questions regarding heritable genome editing, with a focus on: a) the prevention of heritable genetic conditions; b) editing for immunity; and c) editing for enhancement. The aim is to understand the views on these issues so as to inform further research and policy, and to analyse the process and effect of deliberation on opinion. Participants will be expected to study the provided resource materials and pass the entrance exam—aligning with the protocols of the Harvard Personal Genome Project. In this way, the commitment, openness and basic knowledge of the candidates will be tested to ascertain whether they are suitable participants for the deliberative engagement.

Perception of personalized medicine, pharmacogenomics, and genetic testing among undergraduates in Hong Kong

Background The global development and advancement of genomic medicine in the recent decade has accelerated the implementation of personalized medicine (PM) and pharmacogenomics (PGx) into clinical practice, while catalyzing the emergence of genetic testing (GT) with relevant ethical, legal, and social implications (ELSI). Results The perception of university undergraduates with regards to PM and PGx was investigated, and 80% of undergraduates valued PM as a promising healthcare model with 66% indicating awareness of personal genome testing companies. When asked about the curriculum design towards PM and PGx, compared to undergraduates in non-medically related curriculum, those studying in medically related curriculum had an adjusted 7.2 odds of perceiving that their curriculum was well-designed for learning PGx (95% CI 3.6–14.6) and a 3.7 odds of perceiving that PGx was important in their study (95% CI 2.0–6.8). Despite this, only 16% of medically related curriculum undergraduates would consider embarking on future education on PM. When asked about their perceptions on GT, 60% rated their genetic knowledge as “School Biology” level or below while 76% would consider undergoing a genetic test. As for ELSI, 75% of undergraduates perceived that they were aware of ethical issues of GT in general, particularly on “Patient Privacy” (80%) and “Data Confidentiality” (68%). Undergraduates were also asked about their perceived reaction upon receiving an unfavorable result from GT, and over half of the participants perceived that they would feel “helpless or pessimistic” (56%), “inadequate or different” (59%), and “disadvantaged at job seeking” (59%), while older undergraduates had an adjusted 2.0 odds of holding the latter opinion (95% CI 1.1–3.5), compared to younger undergraduates. Conclusion Hong Kong undergraduates showed a high awareness of PM but insufficient genetic knowledge and low interest in pursuing a career towards PM. They were generally aware of ethical issues of GT and especially concerned about patient privacy and data confidentiality. There was a predominance of pessimistic views towards unfavorable testing results. This study calls for the attention to evaluate education and talent development on genomics, and update existing legal frameworks on genetic testing in Hong Kong.