Here, we describe single-tube long fragment read (stLFR), a technology that enables sequencing of data from long DNA molecules using economical second-generation sequencing technology. It is based on ...adding the same barcode sequence to subfragments of the original long DNA molecule (DNA cobarcoding). To achieve this efficiently, stLFR uses the surface of microbeads to create millions of miniaturized barcoding reactions in a single tube. Using a combinatorial process, up to 3.6 billion unique barcode sequences were generated on beads, enabling practically nonredundant cobarcoding with 50 million barcodes per sample. Using stLFR, we demonstrate efficient unique cobarcoding of more than 8 million 20- to 300-kb genomic DNA fragments. Analysis of the human genome NA12878 with stLFR demonstrated high-quality variant calling and phase block lengths up to N50 34 Mb. We also demonstrate detection of complex structural variants and complete diploid de novo assembly of NA12878. These analyses were all performed using single stLFR libraries, and their construction did not significantly add to the time or cost of whole-genome sequencing (WGS) library preparation. stLFR represents an easily automatable solution that enables high-quality sequencing, phasing, SV detection, scaffolding, cost-effective diploid de novo genome assembly, and other long DNA sequencing applications.
There is a rapidly increasing amount of de novo genome assembly using next-generation sequencing (NGS) short reads; however, several big challenges remain to be overcome in order for this to be ...efficient and accurate. SOAPdenovo has been successfully applied to assemble many published genomes, but it still needs improvement in continuity, accuracy and coverage, especially in repeat regions.
To overcome these challenges, we have developed its successor, SOAPdenovo2, which has the advantage of a new algorithm design that reduces memory consumption in graph construction, resolves more repeat regions in contig assembly, increases coverage and length in scaffold construction, improves gap closing, and optimizes for large genome.
Benchmark using the Assemblathon1 and GAGE datasets showed that SOAPdenovo2 greatly surpasses its predecessor SOAPdenovo and is competitive to other assemblers on both assembly length and accuracy. We also provide an updated assembly version of the 2008 Asian (YH) genome using SOAPdenovo2. Here, the contig and scaffold N50 of the YH genome were ~20.9 kbp and ~22 Mbp, respectively, which is 3-fold and 50-fold longer than the first published version. The genome coverage increased from 81.16% to 93.91%, and memory consumption was ~2/3 lower during the point of largest memory consumption.
The boost of next-generation sequencing technologies provides us with an unprecedented opportunity for elucidating genetic mysteries, yet the short-read length hinders us from better assembling the ...genome from scratch. New protocols now exist that can generate overlapping pair-end reads. By joining the 3' ends of each read pair, one is able to construct longer reads for assembling. However, effectively joining two overlapped pair-end reads remains a challenging task.
In this article, we present an efficient tool called Connecting Overlapped Pair-End (COPE) reads, to connect overlapping pair-end reads using k-mer frequencies. We evaluated our tool on 30× simulated pair-end reads from Arabidopsis thaliana with 1% base error. COPE connected over 99% of reads with 98.8% accuracy, which is, respectively, 10 and 2% higher than the recently published tool FLASH. When COPE is applied to real reads for genome assembly, the resulting contigs are found to have fewer errors and give a 14-fold improvement in the N50 measurement when compared with the contigs produced using unconnected reads.
COPE is implemented in C++ and is freely available as open-source code at ftp://ftp.genomics.org.cn/pub/cope.
twlam@cs.hku.hk or luoruibang@genomics.org.cn
Recent advances in long fragment read (LFR, also known as linked-read technologies or read-cloud) technologies, such as single tube long fragment reads (stLFR), 10X Genomics Chromium reads, and ...TruSeq synthetic long-reads, have enabled efficient haplotyping and genome assembly. However, in the case of stLFR and 10X Genomics Chromium reads, the long fragments of a genome are covered sparsely by reads in each barcode and most barcodes are contained in multiple long fragments from different regions, which results in inefficient assembly when using long-range information. Thus, methods to address these shortcomings are vital for capitalizing on the additional information obtained using these technologies. We therefore designed IterCluster, a novel, alignment-free clustering algorithm that can cluster barcodes from the same target region of a genome, using -mer frequency-based features and a Markov Cluster (MCL) approach to identify enough reads in a target region of a genome to ensure sufficient target genome sequence depth. The IterCluster method was validated using BGI stLFR and 10X Genomics chromium reads datasets. IterCluster had a higher precision and recall rate on BGI stLFR data compared to 10X Genomics Chromium read data. In addition, we demonstrated how IterCluster improves the de novo assembly results when using a divide-and-conquer strategy on a human genome data set (scaffold/contig N50 = 13.2 kbp/7.1 kbp vs. 17.1 kbp/11.9 kbp before and after IterCluster, respectively). IterCluster provides a new way for determining LFR barcode enrichment and a novel approach for de novo assembly using LFR data. IterCluster is OpenSource and available on https://github.com/JianCong-WENG/IterCluster.
Background
With the advancement of whole‐genome sequencing (WGS) technology, massively parallel sequencing (MPS) remains the mainstream due to its accuracy, low cost, and high throughput. The ...development of the analytical pipeline corresponding to MPS has always been of great importance. Increasingly large population genomics studies, as a specific type of big data research, pose new challenges for analysis solutions.
Results
Here, we introduce ZBOLT, a comprehensive analysis system that incorporates both software and hardware advancements, making it an appropriate choice for large‐scale population genomic studies that require extensive data processing. In this study, we first evaluate ZBOLT's calling accuracy using the Genome in a Bottle (GIAB) benchmark dataset. Then we apply ZBOLT to a large‐scale population genomics study with 5,616 high sequencing depth samples totaling 1.16Pbp (base pair). As the results show, ZBOLT demonstrates exceptional efficiency and low energy consumption, processing 100Tbp per day and using 1kWh per 100Gbp sequenced sample.
Conclusion
This research serves as a valuable reference for analyzing sequencing data from large population cohorts and underscores the significant potential of ZBOLT in large‐scale population genomics studies.
ZBOLT is a comprehensive analysis system that incorporates both software and hardware advancements, making it an appropriate choice for large‐scale population genomic studies.
ZBOLT's calling accuracy is comparable to GATK Best Practices.
ZBOLT is able to analyze 100Tbp of raw sequencing data per day.
ZBOLT consumes 1 kWh per 100Gbp sequenced sample.
Transcriptome sequencing has long been the favored method for quickly and inexpensively obtaining a large number of gene sequences from an organism with no reference genome. Owing to the rapid ...increase in throughputs and decrease in costs of next- generation sequencing, RNA-Seq in particular has become the method of choice. However, the very short reads (e.g. 2 × 90 bp paired ends) from next generation sequencing makes de novo assembly to recover complete or full-length transcript sequences an algorithmic challenge.
Here, we present SOAPdenovo-Trans, a de novo transcriptome assembler designed specifically for RNA-Seq. We evaluated its performance on transcriptome datasets from rice and mouse. Using as our benchmarks the known transcripts from these well-annotated genomes (sequenced a decade ago), we assessed how SOAPdenovo-Trans and two other popular transcriptome assemblers handled such practical issues as alternative splicing and variable expression levels. Our conclusion is that SOAPdenovo-Trans provides higher contiguity, lower redundancy and faster execution.
Source code and user manual are available at http://sourceforge.net/projects/soapdenovotrans/.
Optogenetic tools enable examination of how specific cell types contribute to brain circuit functions. A long-standing question is whether it is possible to independently activate two distinct neural ...populations in mammalian brain tissue. Such a capability would enable the study of how different synapses or pathways interact to encode information in the brain. Here we describe two channelrhodopsins, Chronos and Chrimson, discovered through sequencing and physiological characterization of opsins from over 100 species of alga. Chrimson's excitation spectrum is red shifted by 45 nm relative to previous channelrhodopsins and can enable experiments in which red light is preferred. We show minimal visual system-mediated behavioral interference when using Chrimson in neurobehavioral studies in Drosophila melanogaster. Chronos has faster kinetics than previous channelrhodopsins yet is effectively more light sensitive. Together these two reagents enable two-color activation of neural spiking and downstream synaptic transmission in independent neural populations without detectable cross-talk in mouse brain slice.
Insects are the most speciose group of animals, but the phylogenetic relationships of many major lineages remain unresolved. We inferred the phylogeny of insects from 1478 protein-coding genes. ...Phylogenomic analyses of nucleotide and amino acid sequences, with site-specific nucleotide or domain-specific amino acid substitution models, produced statistically robust and congruent results resolving previously controversial phylogenetic relationships. We dated the origin of insects to the Early Ordovician ~479 million years ago (Ma), of insect flight to the Early Devonian (~406 Ma), of major extant lineages to the Mississippian (~345 Ma), and the major diversification of holometabolous insects to the Early Cretaceous. Our phylogenomic study provides a comprehensive reliable scaffold for future comparative analyses of evolutionary innovations among insects.
The Pacific oyster Crassostrea gigas belongs to one of the most species-rich but genomically poorly explored phyla, the Mollusca. Here we report the sequencing and assembly of the oyster genome using ...short reads and a fosmid-pooling strategy, along with transcriptomes of development and stress response and the proteome of the shell. The oyster genome is highly polymorphic and rich in repetitive sequences, with some transposable elements still actively shaping variation. Transcriptome studies reveal an extensive set of genes responding to environmental stress. The expansion of genes coding for heat shock protein 70 and inhibitors of apoptosis is probably central to the oyster's adaptation to sessile life in the highly stressful intertidal zone. Our analyses also show that shell formation in molluscs is more complex than currently understood and involves extensive participation of cells and their exosomes. The oyster genome sequence fills a void in our understanding of the Lophotrochozoa.
Many phylogenomic studies based on transcriptomes have been limited to "single-copy" genes due to methodological challenges in homology and orthology inferences. Only a relatively small number of ...studies have explored analyses beyond reconstructing species relationships. We sampled 69 transcriptomes in the hyperdiverse plant clade Caryophyllales and 27 outgroups from annotated genomes across eudicots. Using a combined similarity- and phylogenetic tree-based approach, we recovered 10,960 homolog groups, where each was represented by at least eight ingroup taxa. By decomposing these homolog trees, and taking gene duplications into account, we obtained 17,273 ortholog groups, where each was represented by at least ten ingroup taxa. We reconstructed the species phylogeny using a 1,122-gene data set with a gene occupancy of 92.1%. From the homolog trees, we found that both synonymous and nonsynonymous substitution rates in herbaceous lineages are up to three times as fast as in their woody relatives. This is the first time such a pattern has been shown across thousands of nuclear genes with dense taxon sampling. We also pinpointed regions of the Caryophyllales tree that were characterized by relatively high frequencies of gene duplication, including three previously unrecognized whole-genome duplications. By further combining information from homolog tree topology and synonymous distance between paralog pairs, phylogenetic locations for 13 putative genome duplication events were identified. Genes that experienced the greatest gene family expansion were concentrated among those involved in signal transduction and oxidoreduction, including a cytochrome P450 gene that encodes a key enzyme in the betalain synthesis pathway. Our approach demonstrates a new approach for functional phylogenomic analysis in nonmodel species that is based on homolog groups in addition to inferred ortholog groups.
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