Three amphioxus reference genomes reveal gene and chromosome evolution of chordates.

The slow-evolving invertebrate amphioxus has an irreplaceable role in advancing our understanding of the vertebrate origin and innovations. Here we resolve the nearly complete chromosomal genomes of three amphioxus species, one of which best recapitulates the 17 chordate ancestor linkage groups. We reconstruct the fusions, retention, or rearrangements between descendants of whole-genome duplications, which gave rise to the extant microchromosomes likely existed in the vertebrate ancestor. Similar to vertebrates, the amphioxus genome gradually establishes its three-dimensional chromatin architecture at the onset of zygotic activation and forms two topologically associated domains at the Hox gene cluster. We find that all three amphioxus species have ZW sex chromosomes with little sequence differentiation, and their putative sex-determining regions are nonhomologous to each other. Our results illuminate the unappreciated interspecific diversity and developmental dynamics of amphioxus genomes and provide high-quality references for understanding the mechanisms of chordate functional genome evolution.

Exploration of whole genome amplification generated chimeric sequences in long-read sequencing data.

MOTIVATION: Multiple displacement amplification (MDA) has become the most commonly used method of whole genome amplification, generating a vast amount of DNA with higher molecular weight and greater genome coverage. Coupling with long-read sequencing, it is possible to sequence the amplicons of over 20 kb in length. However, the formation of chimeric sequences (chimeras, expressed as structural errors in sequencing data) in MDA seriously interferes with the bioinformatics analysis but its influence on long-read sequencing data is unknown. RESULTS: We sequenced the phi29 DNA polymerase-mediated MDA amplicons on the PacBio platform and analyzed chimeras within the generated data. The 3rd-ChimeraMiner has been constructed as a pipeline for recognizing and restoring chimeras into the original structures in long-read sequencing data, improving the efficiency of using TGS data. Five long-read datasets and one high-fidelity long-read dataset with various amplification folds were analyzed. The result reveals that the mis-priming events in amplification are more frequently occurring than widely perceived, and the propor tion gradually accumulates from 42% to over 78% as the amplification continues. In total, 99.92% of recognized chimeric sequences were demonstrated to be artifacts, whose structures were wrongly formed in MDA instead of existing in original genomes. By restoring chimeras to their original structures, the vast majority of supplementary alignments that introduce false-positive structural variants are recycled, removing 97% of inversions on average and contributing to the analysis of structural variation in MDA-amplified samples. The impact of chimeras in long-read sequencing data analysis should be emphasized, and the 3rd-ChimeraMiner can help to quantify and reduce the influence of chimeras. AVAILABILITY AND IMPLEMENTATION: The 3rd-ChimeraMiner is available on GitHub, https://github.com/dulunar/3rdChimeraMiner.

Unraveling the complex evolutionary features of the Cinnamomum camphora mitochondrial genome.

KEY MESSAGE: We reported the mitochondrial genome of Cinnamomum camphora for the first time, revealing frequent rearrangement events in the non-coding regions of Magnoliids mitochondrial genomes. As one of the representative species in the Lauraceae family of Magnoliids, Cinnamomum camphora holds significant economic and ecological value. In this study, the mitochondrial genome (mitogenome) of C. camphora was complete assembled and annotated using PacBio HiFi sequencing. The C. camphora mitogenome is characterized by a branch structure, spans 900,894 bp, and contains 43 protein-coding genes (PCGs), 24 tRNAs, and 3 rRNAs. Most of these PCGs are under purifying selection, with only two (ccmFc and rps7) exhibiting signs of positive selection. The C. camphora mitogenome contains numerous repetitive sequences and intracellular gene transfers, with a total of 36 mitochondrial plastid DNAs, amounting to a combined length of 23,816 bp. Comparative analysis revealed that the non-coding regions of Magnoliids mitogenomes have undergone frequent rearrangements during evolution, but the coding sequences remain highly conserved (more than 98% similarity for protein-coding sequences). Furthermore, a maximum-likelihood phylogenetic tree was reconstructed based on 25 PCGs from 23 plant mitogenomes. The analysis supports the closest relationship between C. camphora and C. chekiangense, consistent with the APG IV classification system. This study elucidates the unique evolutionary features of the C. camphora mitogenome, which will provide valuable insights into the study of genetics and evolution of the family Lauraceae.

Assembly and comparative analysis of the first complete mitochondrial genome of Acer truncatum Bunge: a woody oil-tree species producing nervonic acid.

BACKGROUND: Acer truncatum (purpleblow maple) is a woody tree species that produces seeds with high levels of valuable fatty acids (especially nervonic acid). The species is admired as a landscape plant with high developmental prospects and scientific research value. The A. truncatum chloroplast genome has recently been reported; however, the mitochondrial genome (mitogenome) is still unexplored. RESULTS: We characterized the A. truncatum mitogenome, which was assembled using reads from PacBio and Illumina sequencing platforms, performed a comparative analysis against different species of Acer. The circular mitogenome of A. truncatum has a length of 791,052 bp, with a base composition of 27.11% A, 27.21% T, 22.79% G, and 22.89% C. The A. truncatum mitogenome contains 62 genes, including 35 protein-coding genes, 23 tRNA genes and 4 rRNA genes. We also examined codon usage, sequence repeats, RNA editing and selective pressure in the A. truncatum mitogenome. To determine the evolutionary and taxonomic status of A. truncatum, we conducted a phylogenetic analysis based on the mitogenomes of A. truncatum and 25 other taxa. In addition, the gene migration from chloroplast and nuclear genomes to the mitogenome were analyzed. Finally, we developed a novel NAD1 intron indel marker for distinguishing several Acer species. CONCLUSIONS: In this study, we assembled and annotated the mitogenome of A. truncatum, a woody oil-tree species producing nervonic acid. The results of our analyses provide comprehensive information on the A. truncatum mitogenome, which would facilitate evolutionary research and molecular barcoding in Acer.

VGSC2: Second generation vector graph toolkit of genome synteny and collinearity.

Synteny and collinearity analysis is a standard investigative strategy done in many comparative genomic studies to understand genomic conservation and evolution. Currently, most visualization toolkits of synteny and collinearity do not emphasize the graphical representation of the results, especially the lack of extensible format on vector graphics outputs. This limitation becomes more apparent as 3rd generation sequencing brings high-throughput data, requiring relatively higher resolution for the resulting images. We developed VGSC2, the 2nd version of the web-based vector graph toolkit for genome synteny and collinearity analysis. The updated version enables four types of plots for synteny and collinearity, and three types of plots for gene family evolutionary research. Using web-based technologies, VGSC2 provides an easy-to-use user interface to display the homologous genomic result into vector graphs such as SVG, EPS, and PDF, as well as an online editor. VGSC2 is open source and freely available for use online through the web server available at http://bio.njfu.edu.cn/vgsc2.

Characterization and Analysis of the Mitochondrial Genome of Common Bean (Phaseolus vulgaris) by Comparative Genomic Approaches.

The common bean (Phaseolus vulgaris) is a major source of protein and essential nutrients for humans. To explore the genetic diversity and phylogenetic relationships of P. vulgaris, its complete mitochondrial genome (mitogenome) was sequenced and assembled. The mitogenome is 395,516 bp in length, including 31 unique protein-coding genes (PCGs), 15 transfer RNA (tRNA) genes, and 3 ribosomal RNA (rRNA) genes. Among the 31 PCGs, four genes (mttB, nad1, nad4L, and rps10) use ACG as initiation codons, which are altered to standard initiation codons by RNA editing. In addition, the termination codon CGA in the ccmFC gene is converted to UGA. Selective pressure analysis indicates that the ccmB, ccmFC, rps1, rps10, and rps14 genes were under evolutionary positive selection. The proportions of five amino acids (Phe, Leu, Pro, Arg, and Ser) in the whole amino acid profile of the proteins in each mitogenome can be used to distinguish angiosperms from gymnosperms. Phylogenetic analyses show that P. vulgaris is evolutionarily closer to the Glycininae than other leguminous plants. The results of the present study not only provide an important opportunity to conduct further genomic breeding studies in the common bean, they also provide valuable information for future evolutionary and molecular studies of leguminous plants.

ChimeraMiner: An Improved Chimeric Read Detection Pipeline and Its Application in Single Cell Sequencing.

As the most widely-used single cell whole genome amplification (WGA) approach, multiple displacement amplification (MDA) has a superior performance, due to the high-fidelity and processivity of phi29 DNA polymerase. However, chimeric reads, generated in MDA, cause severe disruption in many single-cell studies. Herein, we constructed ChimeraMiner, an improved chimeric read detection pipeline for analyzing the sequencing data of MDA and classified the chimeric sequences. Two datasets (MDA1 and MDA2) were used for evaluating and comparing the efficiency of ChimeraMiner and previous pipeline. Under the same hardware condition, ChimeraMiner spent only 43.4% (43.8% for MDA1 and 43.0% for MDA2) processing time. Respectively, 24.4 million (6.31%) read pairs out of 773 million reads, and 17.5 million (6.62%) read pairs out of 528 million reads were accurately classified as chimeras by ChimeraMiner. In addition to finding 83.60% (17,639,371) chimeras, which were detected by previous pipelines, ChimeraMiner screened 6,736,168 novel chimeras, most of which were missed by the previous pipeline. Applying in single-cell datasets, all three types of chimera were discovered in each dataset, which introduced plenty of false positives in structural variation (SV) detection. The identification and filtration of chimeras by ChimeraMiner removed most of the false positive SVs (83.8%). ChimeraMiner revealed improved efficiency in discovering chimeric reads, and is promising to be widely used in single-cell sequencing.

Obtaining Genome Sequences of Mutualistic Bacteria in Single Microcystis Colonies.

Cells of Microcystis are associated with heterotrophic bacteria and organized in colonies in natural environment, which are basic elements in the mass occurrence of cyanobacterial species. Analyzing these colonies by using metagenomics is helpful to understand species composition and relationship. Meanwhile, the difference in population abundance among Microcystis colonies could be used to recover genome bins from metagenome assemblies. Herein, we designed a pipeline to obtain high-quality genomes of mutualistic bacteria from single natural Microcystis colonies. Single colonies were lysed, and then amplified by using multiple displacement amplification to overcome the DNA quantity limit. A two-step assembly was performed after sequencing and scaffolds were grouped into putative bins based on their differential-coverage among species. We analyzed six natural colonies of three prevailing Microcystis species from Lake Taihu. Clustering results proved that colonies of the same species were similar in the microbial community composition. Eight putative population genome bins with wide bacterial diversity and different GC content were identified based on coverage difference among colonies. At the phylum level, proteobacteria was the most abundant besides cyanobacteria. Six of the population bins were further refined into nearly complete genomes (completeness > 90%).

Phagocytic intracellular digestion in amphioxus (Branchiostoma).

The digestive methods employed by amphioxus (Branchiostoma)-both intracellular phagocytic digestion and extracellular digestion-have been discussed since 1937. Recent studies also show that epithelial cells lining the Branchiostoma digestive tract can express many immune genes. Here, in Branchiostoma belcheri, using a special tissue fixation method, we show that some epithelial cells, especially those lining the large diverticulum protruding from the gut tube, phagocytize food particles directly, and Branchiostoma can rely on this kind of phagocytic intracellular digestion to obtain energy throughout all stages of its life. Gene expression profiles suggest that diverticulum epithelial cells have functional features of both digestive cells and phagocytes. In starved Branchiostoma, these cells accumulate endogenous digestive and hydrolytic enzymes, whereas, when sated, they express many kinds of immune genes in response to stimulation by phagocytized food particles. We also found that the distal hindgut epithelium can phagocytize food particles, but not as many. These results illustrate phagocytic intercellular digestion in Branchiostoma, explain why Branchiostoma digestive tract epithelial cells express typical immune genes and suggest that the main physiological function of the Branchiostoma diverticulum is different from that of the vertebrate liver.

Complete chloroplast genome sequence of a major economic species, Ziziphus jujuba (Rhamnaceae).

Ziziphus jujuba is an important woody plant with high economic and medicinal value. Here, we analyzed and characterized the complete chloroplast (cp) genome of Z. jujuba, the first member of the Rhamnaceae family for which the chloroplast genome sequence has been reported. We also built a web browser for navigating the cp genome of Z. jujuba ( http://bio.njfu.edu.cn/gb2/gbrowse/Ziziphus_jujuba_cp/ ). Sequence analysis showed that this cp genome is 161,466 bp long and has a typical quadripartite structure of large (LSC, 89,120 bp) and small (SSC, 19,348 bp) single-copy regions separated by a pair of inverted repeats (IRs, 26,499 bp). The sequence contained 112 unique genes, including 78 protein-coding genes, 30 transfer RNAs, and four ribosomal RNAs. The genome structure, gene order, GC content, and codon usage are similar to other typical angiosperm cp genomes. A total of 38 tandem repeats, two forward repeats, and three palindromic repeats were detected in the Z. jujuba cp genome. Simple sequence repeat (SSR) analysis revealed that most SSRs were AT-rich. The homopolymer regions in the cp genome of Z. jujuba were verified and manually corrected by Sanger sequencing. One-third of mononucleotide repeats were found to be erroneously sequenced by the 454 pyrosequencing, which resulted in sequences of 1-4 bases shorter than that by the Sanger sequencing. Analyzing the cp genome of Z. jujuba revealed that the IR contraction and expansion events resulted in ycf1 and rps19 pseudogenes. A phylogenetic analysis based on 64 protein-coding genes showed that Z. jujuba was closely related to members of the Elaeagnaceae family, which will be helpful for phylogenetic studies of other Rosales species. The complete cp genome sequence of Z. jujuba will facilitate population, phylogenetic, and cp genetic engineering studies of this economic plant.

The first mitogenome of Lauraceae (Cinnamomum chekiangense).

•The first reported mitochondrial genome (Cinnamomum chekiangense) of the Lauraceae family.•The mitogenome of C. chekiangense retains almost all of the ancestral protein-coding genes and has the highest RNA editing number in angiosperms.•Both of the plastid and mitochondrial phylogenetic trees support the magnoliids as a sister group of monocots and eudicots.

PMAT: an efficient plant mitogenome assembly toolkit using low-coverage HiFi sequencing data.

Complete mitochondrial genomes (mitogenomes) of plants are valuable resources for nucleocytoplasmic interactions, plant evolution, and plant cytoplasmic male sterile line breeding. However, the complete assembly of plant mitogenomes is challenging due to frequent recombination events and horizontal gene transfers. Previous studies have adopted Illumina, PacBio, and Nanopore sequencing data to assemble plant mitogenomes, but the poor assembly completeness, low sequencing accuracy, and high cost limit the sampling capacity. Here, we present an efficient assembly toolkit (PMAT) for de novo assembly of plant mitogenomes using low-coverage HiFi sequencing data. PMAT has been applied to the de novo assembly of 13 broadly representative plant mitogenomes, outperforming existing organelle genome assemblers in terms of assembly accuracy and completeness. By evaluating the assembly of plant mitogenomes from different sequencing data, it was confirmed that PMAT only requires 1× HiFi sequencing data to obtain a complete plant mitogenome. The source code for PMAT is available at https://github.com/bichangwei/PMAT. The developed PMAT toolkit will indeed accelerate the understanding of evolutionary variation and breeding application of plant mitogenomes.

The allotetraploid horseradish genome provides insights into subgenome diversification and formation of critical traits.

Polyploidization can provide a wealth of genetic variation for adaptive evolution and speciation, but understanding the mechanisms of subgenome evolution as well as its dynamics and ultimate consequences remains elusive. Here, we report the telomere-to-telomere (T2T) gap-free reference genome of allotetraploid horseradish (Armoracia rusticana) sequenced using a comprehensive strategy. The (epi)genomic architecture and 3D chromatin structure of the A and B subgenomes differ significantly, suggesting that both the dynamics of the dominant long terminal repeat retrotransposons and DNA methylation have played critical roles in subgenome diversification. Investigation of the genetic basis of biosynthesis of glucosinolates (GSLs) and horseradish peroxidases reveals both the important role of polyploidization and subgenome differentiation in shaping the key traits. Continuous duplication and divergence of essential genes of GSL biosynthesis (e.g., FMOGS-OX, IGMT, and GH1 gene family) contribute to the broad GSL profile in horseradish. Overall, the T2T assembly of the allotetraploid horseradish genome expands our understanding of polyploid genome evolution and provides a fundamental genetic resource for breeding and genetic improvement of horseradish.

Assembly and comparative analysis of the complete mitochondrial genome of Salix wilsonii using PacBio HiFi sequencing.

Salix L. (willows) is one of the most taxonomically complex genera of flowering plants, including shrubs, tall trees, bushes, and prostrate plants. Despite the high species diversity, only five mitochondrial genomes (mitogenomes) have been released in this genus. Salix wilsonii is an important ornamental and economic willow tree in section Wilsonia of the genus Salix. In this study, the S. wilsonii mitogenome was assembled into a typical circular structure with a size of 711,456 bp using PacBio HiFi sequencing. A total of 58 genes were annotated in the S. wilsonii mitogenome, including 33 protein-coding genes (PCGs), 22 tRNAs, and 3 rRNAs. In the S. wilsonii mitogenome, four genes (mttB, nad3, nad4, and sdh4) were found to play important roles in its evolution through selection pressure analysis. Collinearity analysis of six Salix mitogenomes revealed high structural variability. To determine the evolutionary position of S. wilsonii, we conducted a phylogenetic analysis of the mitogenomes of S. wilsonii and 12 other species in the order Malpighiales. Results strongly supported the segregation of S. wilsonii and other five Salix species with 100% bootstrap support. The comparative analysis of the S. wilsonii mitogenome not only sheds light on the functional and structural features of S. wilsonii but also provides essential information for genetic studies of the genus Salix.

Deciphering the Multi-Chromosomal Mitochondrial Genome of Populus simonii.

Mitochondria, inherited maternally, are energy metabolism organelles that generate most of the chemical energy needed to power cellular various biochemical reactions. Deciphering mitochondrial genome (mitogenome) is important for elucidating vital activities of species. The complete chloroplast (cp) and nuclear genome sequences of Populus simonii (P. simonii) have been reported, but there has been little progress in its mitogenome. Here, we assemble the complete P. simonii mitogenome into three circular-mapping molecules (lengths 312.5, 283, and 186 kb) with the total length of 781.5 kb. All three molecules of the P. simonii mitogenome had protein-coding capability. Whole-genome alignment analyses of four Populus species revealed the fission of poplar mitogenome in P. simonii. Comparative repeat analyses of four Populus mitogenomes showed that there were no repeats longer than 350 bp in Populus mitogenomes, contributing to the stability of genome sizes and gene contents in the genus Populus. As the first reported multi-circular mitogenome in Populus, this study of P. simonii mitogenome are imperative for better elucidating their biological functions, replication and recombination mechanisms, and their unique evolutionary trajectories in Populus.

FEM: mining biological meaning from cell level in single-cell RNA sequencing data.

BACKGROUND: One goal of expression data analysis is to discover the biological significance or function of genes that are differentially expressed. Gene Set Enrichment (GSE) analysis is one of the main tools for function mining that has been widely used. However, every gene expressed in a cell is valuable information for GSE for single-cell RNA sequencing (scRNA-SEQ) data and not should be discarded. METHODS: We developed the functional expression matrix (FEM) algorithm to utilize the information from all expressed genes. The algorithm converts the gene expression matrix (GEM) into a FEM. The FEM algorithm can provide insight on the biological significance of a single cell. It can also integrate with GEM for downstream analysis. RESULTS: We found that FEM performed well with cell clustering and cell-type specific function annotation in three datasets (peripheral blood mononuclear cells, human liver, and human pancreas).

PreLnc: An Accurate Tool for Predicting lncRNAs Based on Multiple Features.

Accumulating evidence indicates that long non-coding RNAs (lncRNAs) have certain similarities with messenger RNAs (mRNAs) and are associated with numerous important biological processes, thereby demanding methods to distinguish them. Based on machine learning algorithms, a variety of methods are developed to identify lncRNAs, providing significant basic data support for subsequent studies. However, many tools lack certain scalability, versatility and balance, and some tools rely on genome sequence and annotation. In this paper, we propose a convenient and accurate tool "PreLnc", which uses high-confidence lncRNA and mRNA transcripts to build prediction models through feature selection and classifiers. The false discovery rate (FDR) adjusted P-value and Z-value were used for analyzing the tri-nucleotide composition of transcripts of different species. Conclusions can be drawn from the experiment that there were significant differences in RNA transcripts among plants, which may be related to evolutionary conservation and the fact that plants are under evolutionary pressure for a longer time than animals. Combining with the Pearson correlation coefficient, we use the incremental feature selection (IFS) method and the comparison of multiple classifiers to build the model. Finally, the balanced random forest was used to construct the classifier, and PreLnc obtained 91.09% accuracy for 349,186 transcripts of animals and plants. In addition, by comparing standard performance measurements, PreLnc performed better than other prediction tools.

Whole-genome resequencing reveals the pleistocene temporal dynamics of Branchiostoma belcheri and Branchiostoma floridae populations.

Global climatic fluctuations governed the ancestral demographic histories of species and contributed to place the current population status into a more extensive ecological and evolutionary context. Genetic variations will leave unambiguous signatures in the patterns of intraspecific genetic variation in extant species since the genome of each individual is an imperfect mosaic of the ancestral genomes. Here, we report the genome sequences of 20 Branchiostoma individuals by whole-genome resequencing strategy. We detected over 140 million genomic variations for each Branchiostoma individual. In particular, we applied the pairwise sequentially Markovian coalescent (PSMC) method to estimate the trajectories of changes in the effective population size (N e) of Branchiostoma population during the Pleistocene. We evaluated the threshold of sequencing depth for proper inference of demographic histories using PSMC was ≥25×. The PSMC results highlight the role of historical global climatic fluctuations in the long-term population dynamics of Branchiostoma. The inferred ancestral N e of the Branchiostoma belcheri populations from Zhanjiang and Xiamen (China) seawaters was different in amplitude before the first (mutation rate = 3 × 10-9) or third glaciation (mutation rate = 9 × 10-9) of the Pleistocene, indicating that the two populations most probably started to evolve in isolation in their respective seas after the first or third glaciation of the Pleistocene. A pronounced population bottleneck coinciding with the last glacial maximum was observed in all Branchiostoma individuals, followed by a population expansion occurred during the late Pleistocene. Species that have experienced long-term declines may be especially vulnerable to recent anthropogenic activities. Recently, the industrial pollution and the exploitation of sea sand have destroyed the harmonious living environment of amphioxus species. In the future, we need to protect the habitat of Branchiostoma and make full use of these detected genetic variations to facilitate the functional study of Branchiostoma for adaptation to local environments.

Whole-Genome Resequencing of Twenty Branchiostoma belcheri Individuals Provides a Brand-New Variant Dataset for Branchiostoma.

As the extant representatives of the basal chordate lineage, amphioxi (including the genera Branchiostoma, Asymmetron and Epigonichthys) play important roles in tracing the state of chordate ancestry. Previous studies have reported that members of the Branchiostoma species have similar morphological phenotypic characteristics, but in contrast, there are high levels of genetic polymorphisms in the populations. Here, we resequenced 20 Branchiostomabelcheri genomes to an average depth of approximately 12.5X using the Illumina HiSeq 2000 platform. In this study, over 52 million variations (~12% of the total genome) were detected in the B. belcheri population, and an average of 12.8 million variations (~3% of the total genome) were detected in each individual, confirming that Branchiostoma is one of the most genetically diverse species sequenced to date. Demographic inference analysis highlighted the role of historical global temperature in the long-term population dynamics of Branchiostoma, and revealed a population expansion at the Greenlandian stage of the current geological epoch. We detected 594 Single nucleotide polymorphism and 148 Indels in the Branchiostoma mitochondrial genome, and further analyzed their genetic mutations. A recent study found that the epithelial cells of the digestive tract in Branchiostoma can directly phagocytize food particles and convert them into absorbable nontoxic nutrients using powerful digestive and immune gene groups. In this study, we predicted all potential mutations in intracellular digestion-associated genes. The results showed that most "probably damaging" mutations were related to rare variants (MAF<0.05) involved in strengthening or weakening the intracellular digestive capacity of Branchiostoma. Due to the extremely high number of polymorphisms in the Branchiostoma genome, our analysis with a depth of approximately 12.5X can only be considered a preliminary analysis. However, the novel variant dataset provided here is a valuable resource for further investigation of phagocytic intracellular digestion in Branchiostoma and determination of the phenotypic and genotypic features of Branchiostoma.