Designing efficient randstrobes for sequence similarity analyses.

MOTIVATION: Substrings of length k, commonly referred to as k-mers, play a vital role in sequence analysis. However, k-mers are limited to exact matches between sequences leading to alternative constructs. We recently introduced a class of new constructs, strobemers, that can match across substitutions and smaller insertions and deletions. Randstrobes, the most sensitive strobemer proposed in Sahlin (Effective sequence similarity detection with strobemers. Genome Res 2021a;31:2080-94. https://doi.org/10.1101/gr.275648.121), has been used in several bioinformatics applications such as read classification, short-read mapping, and read overlap detection. Recently, we showed that the more pseudo-random the behavior of the construction (measured in entropy), the more efficient the seeds for sequence similarity analysis. The level of pseudo-randomness depends on the construction operators, but no study has investigated the efficacy. RESULTS: In this study, we introduce novel construction methods, including a Binary Search Tree-based approach that improves time complexity over previous methods. To our knowledge, we are also the first to address biases in construction and design three metrics for measuring bias. Our evaluation shows that our methods have favorable speed and sampling uniformity compared to existing approaches. Lastly, guided by our results, we change the seed construction in strobealign, a short-read mapper, and find that the results change substantially. We suggest combining the two results to improve strobealign's accuracy for the shortest reads in our evaluated datasets. Our evaluation highlights sampling biases that can occur and provides guidance on which operators to use when implementing randstrobes. AVAILABILITY AND IMPLEMENTATION: All methods and evaluation benchmarks are available in a public Github repository at https://github.com/Moein-Karami/RandStrobes. The scripts for running the strobealign analysis are found at https://github.com/NBISweden/strobealign-evaluation.

Clonally heritable gene expression imparts a layer of diversity within cell types.

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.

[177Lu]Lu-DOTAGA.Glu.(FAPi)2 Radionuclide Therapy: a New Treatment Option for Patients with Glioblastoma Multiforme.

In this case report, we present the clinical management of a 52-year-old female patient with a recurrent right temporo-parietal glioblastoma multiforme (GBM). The patient presented with symptoms of headache and loss of balance and recurrence on magnetic resonance imaging (MRI). To evaluate the fibroblast activation protein inhibitor (FAPi) expression in the recurrent lesion, an exploratory [68 Ga]Ga-DOTA.SA.FAPi PET/CT scan was performed. The imaging results revealed FAPi expression in the lesion located in the right temporo-parietal region. Based on the findings of FAPi expression, the patient underwent [177Lu]Lu-DOTAGA.Glu.(FAPi)2 treatment. After completing two cycles of [177Lu]Lu-DOTAGA.Glu.(FAPi)2 therapy, a follow-up [68 Ga]Ga-DOTA.SA.FAPi PET/CT scan was conducted. The post-treatment imaging showed a significant reduction in FAPi uptake and regression in the size of the lesion, as well as a decrease in perilesional edema, as observed on the MRI. Furthermore, the patient experienced an improvement in symptoms and performance status. These results suggest that [68 Ga]Ga-DOTA.SA.FAPi monomer imaging and [177Lu]Lu-DOTAGA.Glu.(FAPi)2 dimer therapeutics hold promise for patients with recurrent GBM when other standard-line therapeutic options have been exhausted. This case highlights the potential of using FAPi-based theranostics in the management of recurrent GBM, providing a potential avenue for personalized treatment in patients who have limited treatment options available.

Therapeutic potential of [177Lu]Lu-DOTAGA-FAPi dimers in metastatic breast cancer patients with limited treatment options: efficacy and safety assessment.

PURPOSE: The upregulation of fibroblast activation protein (FAP) expression has been observed in various cancers, including metastatic breast carcinoma, prompting research into small molecule inhibitors for both diagnostic and therapeutic purposes. While the diagnostic value of PET/CT imaging using 68 Ga- or 18F-labelled FAPi-monomers in breast cancer diagnosis is well-established, there is a significant need for therapeutic analogs. This retrospective study aimed to assess the safety and effectiveness of [177Lu]Lu-DOTAGA.FAPi dimer radionuclide therapy in patients with advanced-stage breast cancer who had previously undergone [68 Ga]Ga-DOTA.SA.FAPi PET/CT scans to confirm the expression of FAP. MATERIALS AND METHODS: Between November 2020 and March 2023, a compassionate treatment approach was utilized to administer [177Lu]Lu-DOTAGA.FAPi dimer radionuclide therapy to heavily pretreated patients with advanced breast cancer. Nineteen patients (18 females, 1 male) with metastatic breast cancer participated in the study, with an average age of 44.6 ± 10.7 years. The therapy was administered at intervals of 8 to 12 weeks, and the median follow-up duration was 14 months. The primary objective of the study was to assess molecular response using [68 Ga]Ga-DOTA.SA.FAPi PET/CT scans, with response evaluation based on the PERCIST criteria. Secondary endpoints included overall survival (OS), progression-free survival (PFS), clinical response assessment, and safety evaluation using CTCAE v5.0 guidelines. RESULTS: A total of 65 cycles were administered, with a mean cumulative activity of 19 ± 5.7 GBq (510 ± 154 mCi) ranging from 11 to 33.3 GBq (300 to 900 mCi) of [177Lu]Lu-DOTAGA.FAPi dimer. The number of cycles ranged from 2 to 6, with a median of 3 cycles. The treatment protocol consisted of different numbers of cycles administered to the patients: specifically, two cycles were given to five patients, three cycles to nine patients, four cycles to one patient, and six cycles to four patients. Most patients had invasive/infiltrative ductal carcinoma (94.7%), while a small percentage had invasive lobular carcinoma (5.3%). All patients had bone metastases, and five of them also had liver involvement, while seven had brain metastases. Response assessment using [68 Ga]Ga-DOTA.SA.FAPi PET/CT scans showed that 25% of the 16 patients evaluated had partial remission, while 37.5% exhibited disease progression. According to the VAS response criteria, 26.3% achieved complete response, 15.7% had partial response, 42% showed minimal response, 11% had stable disease, and 5% had no response. The clinical disease control rate was promising, with 95% of patients achieving disease control. The clinical objective response rate was 84%. The median follow-up period was 14 months. At the time of analysis, the median overall survival was 12 months, and the median progression-free survival was 8.5 months. Notably, no severe hematological, renal, or hepatic toxicities, electrolyte imbalances, or adverse events of grade 3 or 4 were observed during the study. CONCLUSION: The findings suggest that [177Lu]Lu-DOTAGA.FAPi dimer therapy is well-tolerated, safe, and effective for treating end-stage metastatic breast cancer patients. [177Lu]Lu-DOTAGA.FAPi dimer treatment demonstrated promising efficacy in patients with advanced breast cancer, as indicated by high disease control rates, favorable response outcomes, and acceptable safety profile. Further research and longer follow-up are warranted to assess long-term outcomes and validate these findings.

BLR: a flexible pipeline for haplotype analysis of multiple linked-read technologies.

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.

Head-to-head comparison of [68Ga]Ga-DOTA.SA.FAPi with [18F]F-FDG PET/CT in radioiodine-resistant follicular-cell derived thyroid cancers.

PURPOSE: In the context of radioiodine-resistant follicular-cell derived thyroid cancers (RAI-R-FCTC), [18F]F-FDG PET/CT serves as a widely used and valuable diagnostic imaging method. However, there is growing interest in utilizing molecular imaging probes that target cancer-associated fibroblasts (CAFs) as an alternative approach. This study sought to compare the diagnostic capabilities of [68Ga]Ga-DOTA.SA.FAPi and [18F]F-FDG PET/CT in patients with RAI-R-FCTC. METHODS: In this retrospective study, a total of 117 patients with RAI-R-FCTC were included. The study population consisted of 68 females and 49 males, with a mean age of 53.2 ± 11.7 years. The aim of the study was to perform a comprehensive qualitative and quantitative assessment of [68Ga]Ga-DOTA.SA.FAPi and [18F]F-FDG PET/CT scans in RAI-R-FCTC patients. The qualitative assessment involved comparing patient-based and lesion-based visual interpretations of both scans, while the quantitative assessment included analyzing standardized uptake values corrected for lean body mass (SULpeak and SULavg). The findings obtained from the scans were validated by correlating them with morphological findings from diagnostic computed tomography and/or histopathological examination. RESULTS: Among the 117 RAI-R-FCTC patients, 60 had unilateral local disease, and 9 had bilateral lesions with complete concordance in the detection rate on both PET scans. [68Ga]Ga-DOTA.SA.FAPi had a higher detection rate for lymph nodes (95.4% vs 86.6%, p<0.0001), liver metastases (100% vs. 81.3%, p<0.0001), and brain metastases (100% vs. 39%, p<0.0001) compared to [18F]F-FDG. The detection rates for pleural and bone metastases were similar between the two radiotracers. For lung metastases, [68Ga]Ga-DOTA.SA.FAPi showed a detection rate of 81.7%, whereas [18F]F-FDG had a detection rate of 64.6%. Remarkably, [68Ga]Ga-DOTA.SA.FAPi was able to detect a bowel metastasis that was missed on [18F]F-FDG scan. The median standardized uptake values (SUL) were generally comparable between the two radiotracers, except for brain metastases (SULpeak [68Ga]Ga-DOTA.SA.FAPi vs. [18F]F-FDG: 13.9 vs. 6.7, p-0.0001) and muscle metastases (SULpeak [68Ga]Ga-DOTA.SA.FAPi vs. [18F]F-FDG: 9.56 vs. 5.62, p-0.0085), where [68Ga]Ga-DOTA.SA.FAPi exhibited higher uptake. CONCLUSION: The study results demonstrate the superior performance of [68Ga]Ga-DOTA.SA.FAPi compared to [18F]F-FDG PET/CT in detecting lymph nodal, liver, bowel, and brain metastases in patients with RAI-R-FCTC. These findings highlight the potential of [68Ga]Ga-DOTA.SA.FAPi as a theranostic tool that can complement the benefits of [18F]F-FDG PET/CT in the imaging of RAI-R-FCTC.

Head-to-Head Comparison of [68Ga]Ga-DOTA.SA.FAPi and [68Ga]Ga-DOTANOC Positron Emission Tomography/Computed Tomography Imaging for the Follow-Up Surveillance of Patients with Medullary Thyroid Cancer.

Background: A theranostic probe for accurate staging and treatment is crucial for the management of medullary thyroid cancers (MTCs). The abundance of stroma in most of thyroid cancers, including MTC, opens new avenues for selecting cancer-associated fibroblasts (CAFs) as new molecular imaging and therapeutic targets. [68Ga]Ga-labeled fibroblast activation protein inhibitor (FAPi) molecules have gained importance as alternative molecular imaging agents in the imaging of thyroid cancers. The purpose of this study was to compare the detection efficiency of primary and metastatic lesions of MTCs between [68Ga]Ga-DOTA.SA.FAPi and [68Ga]Ga-DOTANOC positron emission tomography (PET) radiotracers. Materials and Methods: In this retrospective study, [68Ga]Ga-DOTANOC and [68Ga]Ga-DOTA.SA.FAPi PET/CT (computed tomography) images were compared using patient-based and lesion-based analysis in patients with MTC for follow-up assessment. The quantitative assessment included comparing standardized uptake values corrected for lean body mass (SULpeak) and tumor-to-background ratios (TBR). The findings on both scans were validated with the morphological findings of the diagnostic CT. Results: Twenty-seven patients (21 males and 6 females) with a mean age of 42.4 ± 13.2 years (range 14-66 years) were included in the study. [68Ga]Ga-DOTA.SA.FAPi had similar sensitivities as that of [68Ga]Ga-DOTANOC PET/CT for detecting primary tumors (100% [18 of 18] vs. 94.4% [17 of 18], p = 0.979) involved lymph nodes (98.3% [118 of 120] vs. 95% [114 of 120], p = 0.288), and brain metastases (100%). [68Ga]Ga-DOTA.SA.FAPi demonstrated significantly higher sensitivities than [68Ga]Ga-DOTANOC PET/CT for detecting lung nodules (93.5% [87 of 93] vs. 68.9% [64 of 93], p < 0.0001), liver (100% [105 of 105] vs. 46.4% [49 of 105], p < 0.0001), bone (92.4% [110 of 119] vs. 76.5% [91 of 119], p = 0.001), and pleural metastases 98.2% versus 0%. Higher uptake values and TBR values were reported with [68Ga]Ga-DOTA.SA.FAPi compared with that of [68Ga]Ga-DOTANOC. Conclusion: [68Ga]Ga-DOTA.SA.FAPi outperformed [68Ga]Ga-DOTANOC PET/CT in the detection of distant metastases with both patient-based and lesion-based analysis in MTCs.

Head-to-Head Comparison between [68Ga]Ga-DOTA.SA.FAPi and [18F]F-FDG PET/CT Imaging in Patients with Breast Cancer.

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.

Novel Generation of FAP Inhibitor-Based Homodimers for Improved Application in Radiotheranostics.

Radiopharmaceuticals based on the highly potent FAP inhibitor (FAPi) UAMC-1110 have shown great potential in molecular imaging, but the short tumor retention time of the monomers do not match the physical half-lives of the important therapeutic radionuclides 177Lu and 225Ac. This was improved with the dimer DOTAGA.(SA.FAPi)2, but pharmacological and radiolabeling properties still need optimization. Therefore, the novel FAPi homodimers DO3A.Glu.(FAPi)2 and DOTAGA.Glu.(FAPi)2. were synthesized and quantitatively radiolabeled with 68Ga, 90Y, 177Lu and 225Ac. The radiolabeled complexes showed high hydrophilicity and were generally stable in human serum (HS) and phosphate-buffered saline (PBS) at 37 °C over two half-lives, except for [225Ac]Ac-DOTAGA.Glu.(FAPi)2 in PBS. In vitro affinity studies resulted in subnanomolar IC50 values for FAP and high selectivity for FAP over the related proteases PREP and DPP4 for both compounds as well as for [natLu]Lu-DOTAGA.Glu.(FAPi)2. In a first proof-of-principle patient study (medullary thyroid cancer), [177Lu]Lu-DOTAGA.Glu.(FAPi)2 was compared to [177Lu]Lu-DOTAGA.(SA.FAPi)2. High uptake and long tumor retention was observed in both cases, but [177Lu]Lu-DOTAGA.Glu.(FAPi)2 significantly reduces uptake in non-target and critical organs (liver, colon). Overall, the novel FAPi homodimer DOTAGA.Glu.(FAPi)2 showed improved radiolabeling in vitro and pharmacological properties in vivo compared to DOTAGA.(SA.FAPi)2. [177Lu]Lu-DOTAGA.Glu.(FAPi)2 and [225Ac]Ac-DOTAGA.Glu.(FAPi)2 appear promising for translational application in patients.

Read-Based Phasing and Analysis of Phased Variants with WhatsHap.

WhatsHap is a command-line tool for phasing and phasing-related tasks. It allows to infer haplotypes in diploid and polyploid samples based on (preferably long) reads covering at least two heterozygous variants. It offers additional tools for working with phased variant calls such as computing statistics, comparing different phasings and assigning reads in alignment files to their haplotype.

Clonal relations in the mouse brain revealed by single-cell and spatial transcriptomics.

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.

Characterizing robotic surgical expertise: An exploratory study of neural activation during mental imagery of robotic suturing.

BACKGROUND: Mental imagery (MI) aids skill acquisition, however, it is unclear to what extend MI is used by experienced surgeons. The purpose of this study was to assess differences in MI of participants with varying surgical expertise in robotic surgery. METHODS: Students, residents, and surgeons completed the Mental Imagery Questionnaire to assess MI for robotic suturing. Participants then completed robotic simulator tasks, and imagined performing robotic suturing while being assessed with electroencephalogram (EEG). RESULTS: Attending surgeons reported higher MI for robotic suturing, and EEG revealed higher neural activation during imagery of robotic suturing than other groups. CONCLUSIONS: Experienced surgeons displayed higher MI ability for robotic suturing, and displayed higher cortical activity in the frontal and parietal areas of the brain, which is associated with more advanced motor imagery. MI appears to be a component of robotic surgery expertise.

Sarek: A portable workflow for whole-genome sequencing analysis of germline and somatic variants.

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/.

Extensive dissemination and intraclonal maturation of HIV Env vaccine-induced B cell responses.

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.

From cytogenetics to cytogenomics: whole-genome sequencing as a first-line test comprehensively captures the diverse spectrum of disease-causing genetic variation underlying intellectual disability.

BACKGROUND: 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. METHODS: We analyzed three cohorts with short-read WGS: (i) a retrospective cohort with validated copy number variants (CNVs) (cohort 1, n = 68), (ii) individuals referred for monogenic multi-gene panels (cohort 2, n = 156), and (iii) 100 prospective, consecutive cases referred to our center for CMA (cohort 3). Bioinformatic tools developed include FindSV, SVDB, Rhocall, Rhoviz, and vcf2cytosure. RESULTS: First, we validated our structural variant (SV)-calling pipeline on cohort 1, consisting of three trisomies and 79 deletions and duplications with a median size of 850 kb (min 500 bp, max 155 Mb). All variants were detected. Second, we utilized the same pipeline in cohort 2 and analyzed with monogenic WGS panels, increasing the diagnostic yield to 8%. Next, cohort 3 was analyzed by both CMA and WGS. The WGS data was processed for large (> 10 kb) SVs genome-wide and for exonic SVs and SNVs in a panel of 887 genes linked to intellectual disability as well as genes matched to patient-specific Human Phenotype Ontology (HPO) phenotypes. This yielded a total of 25 pathogenic variants (SNVs or SVs), of which 12 were detected by CMA as well. We also applied short tandem repeat (STR) expansion detection and discovered one pathologic expansion in ATXN7. Finally, a case of Prader-Willi syndrome with uniparental disomy (UPD) was validated in the WGS data. Important positional information was obtained in all cohorts. Remarkably, 7% of the analyzed cases harbored complex structural variants, as exemplified by a ring chromosome and two duplications found to be an insertional translocation and part of a cryptic unbalanced translocation, respectively. CONCLUSION: The overall diagnostic rate of 27% was more than doubled compared to clinical microarray (12%). Using WGS, we detected a wide range of SVs with high accuracy. Since the WGS data also allowed for analysis of SNVs, UPD, and STRs, it represents a powerful comprehensive genetic test in a clinical diagnostic laboratory setting.

High-Quality Library Preparation for NGS-Based Immunoglobulin Germline Gene Inference and Repertoire Expression Analysis.

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.

De Novo Assembly of Two Swedish Genomes Reveals Missing Segments from the Human GRCh38 Reference and Improves Variant Calling of Population-Scale Sequencing Data.

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.

Corrigendum: Cloche is a bHLH-PAS transcription factor that drives haemato-vascular specification.

This corrects the article DOI: 10.1038/nature18614.

Atropos: specific, sensitive, and speedy trimming of sequencing reads.

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.

SweGen: a whole-genome data resource of genetic variability in a cross-section of the Swedish population.

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.