The current human reference sequence (GRCh38) is a foundation for large-scale sequencing projects. However, recent studies have suggested that GRCh38 may be incomplete and give a suboptimal representation of specific population groups. Here, we performed a de novo assembly of two Swedish genomes that revealed over 10 Mb of sequences absent from the human GRCh38 reference in each individual. Around 6 Mb of these novel sequences (NS) are shared with a Chinese personal genome. The NS are highly repetitive, have an elevated GC-content, and are primarily located in centromeric or telomeric regions. Up to 1 Mb of NS can be assigned to chromosome Y, and large segments are also missing from GRCh38 at chromosomes 14, 17, and 21. Inclusion of NS into the GRCh38 reference radically improves the alignment and variant calling from short-read whole-genome sequencing data at several genomic loci. A re-analysis of a Swedish population-scale sequencing project yields > 75,000 putative novel single nucleotide variants (SNVs) and removes > 10,000 false positive SNV calls per individual, some of which are located in protein coding regions. Our results highlight that the GRCh38 reference is not yet complete and demonstrate that personal genome assemblies from local populations can improve the analysis of short-read whole-genome sequencing data.

Well-ordered HIV-1 envelope glycoprotein (Env) trimers are prioritized for clinical evaluation, and there is a need for an improved understanding about how elicited B cell responses evolve following immunization. To accomplish this, we prime-boosted rhesus macaques with clade C NFL trimers and identified 180 unique Ab lineages from ∼1,000 single-sorted Env-specific memory B cells. We traced all lineages in high-throughput heavy chain (HC) repertoire (Rep-seq) data generated from multiple immune compartments and time points and expressed several as monoclonal Abs (mAbs). Our results revealed broad dissemination and high levels of somatic hypermutation (SHM) of most lineages, including tier 2 virus neutralizing lineages, following boosting. SHM was highest in the Ab complementarity determining regions (CDRs) but also surprisingly high in the framework regions (FRs), especially FR3. Our results demonstrate the capacity of the immune system to affinity-mature large numbers of Env-specific B cell lineages simultaneously, supporting the use of regimens consisting of repeated boosts to improve each Ab, even those belonging to less expanded lineages.

Recent advances in sequencing technology have created unprecedented opportunities for biological research. However, the increasing throughput of these technologies has created many challenges for data management and analysis. As the demand for sophisticated analyses increases, the development time of software and algorithms is outpacing the speed of traditional publication. As technologies continue to be developed, methods change rapidly, making publications less relevant for users. The SEQanswers wiki (SEQwiki) is a wiki database that is actively edited and updated by the members of the SEQanswers community (http://SEQanswers.com/). The wiki provides an extensive catalogue of tools, technologies and tutorials for high-throughput sequencing (HTS), including information about HTS service providers. It has been implemented in MediaWiki with the Semantic MediaWiki and Semantic Forms extensions to collect structured data, providing powerful navigation and reporting features. Within 2 years, the community has created pages for over 500 tools, with approximately 400 literature references and 600 web links. This collaborative effort has made SEQwiki the most comprehensive database of HTS tools anywhere on the web. The wiki includes task-focused mini-reviews of commonly used tools, and a growing collection of more than 100 HTS service providers. SEQwiki is available at: http://wiki.SEQanswers.com/.

Ecological adaptation is of major relevance to speciation and sustainable population management, but the underlying genetic factors are typically hard to study in natural populations due to genetic differentiation caused by natural selection being confounded with genetic drift in subdivided populations. Here, we use whole genome population sequencing of Atlantic and Baltic herring to reveal the underlying genetic architecture at an unprecedented detailed resolution for both adaptation to a new niche environment and timing of reproduction. We identify almost 500 independent loci associated with a recent niche expansion from marine (Atlantic Ocean) to brackish waters (Baltic Sea), and more than 100 independent loci showing genetic differentiation between spring- and autumn-spawning populations irrespective of geographic origin. Our results show that both coding and non-coding changes contribute to adaptation. Haplotype blocks, often spanning multiple genes and maintained by selection, are associated with genetic differentiation.

Using both high-throughput sequencing and real-time PCR, the miRNA transcriptome can be analyzed in complementary ways. We describe the necessary bioinformatics pipeline, including software tools, and key methodological steps in the process, such as adapter removal, read mapping, normalization, and multiple testing issues for biomarker identification. The methods are exemplified by the analysis of five favorable (event-free survival) vs. five unfavorable (died of disease) neuroblastoma tumor samples with a total of over 188 million reads.

phiC31 integrase is a sequence-specific phage recombinase that can recombine two short DNA sequences called attB and attP. The enzyme can also promote genomic integration of plasmids carrying attB into native mammalian sequences having partial identity to attP. To increase the efficiency of integration, we mutated the phiC31 integrase gene and screened the mutants in human cells in an assay for higher recombination frequency between attB and attP. We report in this article the isolation of a mutant, P2 that has twice the chromosomal integration frequency of wild-type phiC31 integrase, at both a preintegrated chromosomal attP site and at endogenous pseudo attP sequences in cultured human cells. In mouse liver, P2-mediated integration provided therapeutic long-term levels of human factor IX that were double those generated by wild-type phiC31 integrase. We also describe an additional mutant, P3 that combines the mutations of P2 with further changes and possesses an elevated specificity for integration at a chromosomally placed attP site in human cells. Forty-four percent of colonies carrying integration events mediated by P3 have integration at the placed attP site. These mutant integrases are useful for gene therapy and genome modification, and they demonstrate the feasibility of engineering phiC31 integrase toward more desirable properties.

Whole-genome sequencing (WGS) is a fundamental technology for research to advance precision medicine, but the limited availability of portable and user-friendly workflows for WGS analyses poses a major challenge for many research groups and hampers scientific progress. Here we present Sarek, an open-source workflow to detect germline variants and somatic mutations based on sequencing data from WGS, whole-exome sequencing (WES), or gene panels. Sarek features (i) easy installation, (ii) robust portability across different computer environments, (iii) comprehensive documentation, (iv) transparent and easy-to-read code, and (v) extensive quality metrics reporting. Sarek is implemented in the Nextflow workflow language and supports both Docker and Singularity containers as well as Conda environments, making it ideal for easy deployment on any POSIX-compatible computers and cloud compute environments. Sarek follows the GATK best-practice recommendations for read alignment and pre-processing, and includes a wide range of software for the identification and annotation of germline and somatic single-nucleotide variants, insertion and deletion variants, structural variants, tumour sample purity, and variations in ploidy and copy number. Sarek offers easy, efficient, and reproducible WGS analyses, and can readily be used both as a production workflow at sequencing facilities and as a powerful stand-alone tool for individual research groups. The Sarek source code, documentation and installation instructions are freely available at https://github.com/nf-core/sarek and at https://nf-co.re/sarek/.

The mammalian brain contains many specialized cells that develop from a thin sheet of neuroepithelial progenitor cells. Single-cell transcriptomics revealed hundreds of molecularly diverse cell types in the nervous system, but the lineage relationships between mature cell types and progenitor cells are not well understood. Here we show in vivo barcoding of early progenitors to simultaneously profile cell phenotypes and clonal relations in the mouse brain using single-cell and spatial transcriptomics. By reconstructing thousands of clones, we discovered fate-restricted progenitor cells in the mouse hippocampal neuroepithelium and show that microglia are derived from few primitive myeloid precursors that massively expand to generate widely dispersed progeny. We combined spatial transcriptomics with clonal barcoding and disentangled migration patterns of clonally related cells in densely labeled tissue sections. Our approach enables high-throughput dense reconstruction of cell phenotypes and clonal relations at the single-cell and tissue level in individual animals and provides an integrated approach for understanding tissue architecture.

Linked-read sequencing promises a one-method approach for genome-wide insights including single nucleotide variants (SNVs), structural variants, and haplotyping. We introduce Barcode Linked Reads (BLR), an open-source haplotyping pipeline capable of handling millions of barcodes and data from multiple linked-read technologies including DBS, 10× Genomics, TELL-seq and stLFR. Running BLR on DBS linked-reads yielded megabase-scale phasing with low (<0.2%) switch error rates. Of 13616 protein-coding genes phased in the GIAB benchmark set (v4.2.1), 98.6% matched the BLR phasing. In addition, large structural variants showed concordance with HPRC-HG002 reference assembly calls. Compared to diploid assembly with PacBio HiFi reads, BLR phasing was more continuous when considering switch errors. We further show that integrating long reads at low coverage (∼10×) can improve phasing contiguity and reduce switch errors in tandem repeats. When compared to Long Ranger on 10× Genomics data, BLR showed an increase in phase block N50 with low switch-error rates. For TELL-Seq and stLFR linked reads, BLR generated longer or similar phase block lengths and low switch error rates compared to results presented in the original publications. In conclusion, BLR provides a flexible workflow for comprehensive haplotype analysis of linked reads from multiple platforms.

Cell types can be classified according to shared patterns of transcription. Non-genetic variability among individual cells of the same type has been ascribed to stochastic transcriptional bursting and transient cell states. Using high-coverage single-cell RNA profiling, we asked whether long-term, heritable differences in gene expression can impart diversity within cells of the same type. Studying clonal human lymphocytes and mouse brain cells, we uncovered a vast diversity of heritable gene expression patterns among different clones of cells of the same type in vivo. We combined chromatin accessibility and RNA profiling on different lymphocyte clones to reveal thousands of regulatory regions exhibiting interclonal variation, which could be directly linked to interclonal variation in gene expression. Our findings identify a source of cellular diversity, which may have important implications for how cellular populations are shaped by selective processes in development, aging, and disease. A record of this paper's transparent peer review process is included in the supplemental information.

Comprehensive knowledge of immunoglobulin genetics is required to advance our understanding of B cell biology. Validated immunoglobulin variable (V) gene databases are close to completion only for human and mouse. We present a novel computational approach, IgDiscover, that identifies germline V genes from expressed repertoires to a specificity of 100%. IgDiscover uses a cluster identification process to produce candidate sequences that, once filtered, results in individualized germline V gene databases. IgDiscover was tested in multiple species, validated by genomic cloning and cross library comparisons and produces comprehensive gene databases even where limited genomic sequence is available. IgDiscover analysis of the allelic content of the Indian and Chinese-origin rhesus macaques reveals high levels of immunoglobulin gene diversity in this species. Further, we describe a novel human IGHV3-21 allele and confirm significant gene differences between Balb/c and C57BL6 mouse strains, demonstrating the power of IgDiscover as a germline V gene discovery tool.

Elevated sequencing error rates are the most predominant obstacle in single-nucleotide polymorphism (SNP) detection, which is a major goal in the bulk of current studies using next-generation sequencing (NGS). Beyond routinely handled generic sources of errors, certain base calling errors relate to specific sequence patterns. Statistically principled ways to associate sequence patterns with base calling errors have not been previously described. Extant approaches either incur decisive losses in power, due to relating errors with individual genomic positions rather than motifs, or do not properly distinguish between motif-induced and sequence-unspecific sources of errors.

Mutations in EFTUD2 were proven to cause a very distinct mandibulofacial dysostosis type Guion-Almeida (MFDGA, OMIM #610536). Recently, gross deletions and mutations in EFTUD2 were determined to cause syndromic esophageal atresia (EA), as well. We set forth to find further conditions caused by mutations in the EFTUD2 gene (OMIM *603892).

A key step in the transformation of raw sequencing reads into biological insights is the trimming of adapter sequences and low-quality bases. Read trimming has been shown to increase the quality and reliability while decreasing the computational requirements of downstream analyses. Many read trimming software tools are available; however, no tool simultaneously provides the accuracy, computational efficiency, and feature set required to handle the types and volumes of data generated in modern sequencing-based experiments. Here we introduce Atropos and show that it trims reads with high sensitivity and specificity while maintaining leading-edge speed. Compared to other state-of-the-art read trimming tools, Atropos achieves significant increases in trimming accuracy while remaining competitive in execution times. Furthermore, Atropos maintains high accuracy even when trimming data with elevated rates of sequencing errors. The accuracy, high performance, and broad feature set offered by Atropos makes it an appropriate choice for the pre-processing of Illumina, ABI SOLiD, and other current-generation short-read sequencing datasets. Atropos is open source and free software written in Python (3.3+) and available at https://github.com/jdidion/atropos.

Next generation sequencing (NGS) of immunoglobulin (Ig) repertoires (Rep-seq) enables examination of the adaptive immune system at an unprecedented level. Applications include studies of expressed repertoires, gene usage, somatic hypermutation levels, Ig lineage tracing and identification of genetic variation within the Ig loci through inference methods. All these applications require starting libraries that allow the generation of sequence data with low error rate and optimal representation of the expressed repertoire. Here, we provide detailed protocols for the production of libraries suitable for human Ig germline gene inference and Ig repertoire studies. Various parameters used in the process were tested in order to demonstrate factors that are critical to obtain high quality libraries. We demonstrate an improved 5'RACE technique that reduces the length constraints of Illumina MiSeq based Rep-seq analysis but allows for the acquisition of sequences upstream of Ig V genes, useful for primer design. We then describe a 5' multiplex method for library preparation, which yields full length V(D)J sequences suitable for genotype identification and novel gene inference. We provide comprehensive sets of primers targeting IGHV, IGKV, and IGLV genes. Using the optimized protocol, we produced IgM, IgG, IgK, and IgL libraries and analyzed them using the germline inference tool IgDiscover to identify expressed germline V alleles. This process additionally uncovered three IGHV, one IGKV, and six IGLV novel alleles in a single individual, which are absent from the IMGT reference database, highlighting the need for further study of Ig genetic variation. The library generation protocols presented here enable a robust means of analyzing expressed Ig repertoires, identifying novel alleles and producing individualized germline gene databases from humans.

Since different types of genetic variants, from single nucleotide variants (SNVs) to large chromosomal rearrangements, underlie intellectual disability, we evaluated the use of whole-genome sequencing (WGS) rather than chromosomal microarray analysis (CMA) as a first-line genetic diagnostic test.

This study aimed to compare the diagnostic performance of [68Ga]Ga-DOTA.SA.FAPi with that of [18F]F-FDG PET/CT in detecting primary and metastatic lesions of breast cancer. [18F]F-FDG and [68Ga]Ga-DOTA.SA.FAPi PET/CT scans of histologically proven breast cancer patients were compared according to patient-based and lesion-based analysis. Forty-seven patients with a mean age of 44.8 ± 9.9 years (range: 31-66 years) were evaluated. A total of 85% of patients had invasive ductal carcinoma, and 15% had invasive lobular carcinoma. The tracer uptake [SULpeak, SULavg, and the median tumor-to-background ratio (TBR)] was significantly higher in [68Ga]Ga-DOTA.SA.FAPi than with [18F]F-FDG PET/CT for lymph nodes, pleural metastases, and liver lesions (p < 0.05). However, for brain metastasis, only the median TBR was significantly higher (p < 0.05) compared to [18F]F-FDG. In patient-based analysis the sensitivity of [68Ga]Ga-DOTA.SA.FAPi PET/CT was higher, but not significant than that of [18F]F-FDG PET/CT in the detection of both primary tumors and metastatic lesions. According to lesion-based analysis, on diagnostic CT, 47 patients had 44 primary tumors, 248 lymph nodes, 15 pleural, 88 liver, and 42 brain metastases. [68Ga]Ga-DOTA.SA.FAPi scan identified more abnormal lesions than [18F]F-FDG in all the primary and metastatic sites with a maximum marked difference in the primary site [88.6% vs. 81.8%; p-0.001], lymph nodes [89.1% vs. 83.8%; p-0.0001], pleural metastases [93.3% vs. 73%; p-0.096] and brain metastasis [100% vs. 59.5%; p-0.0001]. [68Ga]Ga-DOTA.SA.FAPi PET/CT was superior to [18F]F-FDG PET/CT in the imaging of breast cancers.

Here we describe the SweGen data set, a comprehensive map of genetic variation in the Swedish population. These data represent a basic resource for clinical genetics laboratories as well as for sequencing-based association studies by providing information on genetic variant frequencies in a cohort that is well matched to national patient cohorts. To select samples for this study, we first examined the genetic structure of the Swedish population using high-density SNP-array data from a nation-wide cohort of over 10 000 Swedish-born individuals included in the Swedish Twin Registry. A total of 1000 individuals, reflecting a cross-section of the population and capturing the main genetic structure, were selected for whole-genome sequencing. Analysis pipelines were developed for automated alignment, variant calling and quality control of the sequencing data. This resulted in a genome-wide collection of aggregated variant frequencies in the Swedish population that we have made available to the scientific community through the website https://swefreq.nbis.se. A total of 29.2 million single-nucleotide variants and 3.8 million indels were detected in the 1000 samples, with 9.9 million of these variants not present in current databases. Each sample contributed with an average of 7199 individual-specific variants. In addition, an average of 8645 larger structural variants (SVs) were detected per individual, and we demonstrate that the population frequencies of these SVs can be used for efficient filtering analyses. Finally, our results show that the genetic diversity within Sweden is substantial compared with the diversity among continental European populations, underscoring the relevance of establishing a local reference data set.