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Abstract For any genome-based research, a robust genome assembly is required. De novo assembly strategies have evolved with changes in DNA sequencing technologies and have been through at least three phases: i) short-read only, ii) short- and long-read hybrid, and iii) long-read only assemblies. Each of the phases has their own error model. We hypothesized that hidden scaffolding errors in short-read assembly and erroneous long-read contigs degrades the quality of short- and long-read hybrid assemblies. We assembled the genome of T. borchgrevinki from data generated during each of the three phases and assessed the quality problems we encountered. We developed strategies such as k-mer-assembled region replacement, parameter optimization, and long-read sampling to address the error models. We demonstrated that a k-mer based strategy improved short-read assemblies as measured by BUSCO while mate-pair libraries introduced hidden scaffolding errors and perturbed BUSCO scores. Further, we found that although hybrid assemblies can generate higher contiguity they tend to suffer from lower quality. In addition, we found long-read only assemblies can be optimized for contiguity by sub-sampling length-restricted raw reads. Our results indicate that long-read contig assembly is the current best choice and that assemblies from phase I and phase II were of lower quality.
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Sequencing Strategy to Ensure Accurate Plasmid Assembly
Despite the wide use of plasmids in research and clinical production, the need to verify plasmid sequences is a bottleneck that is too often underestimated in the manufacturing process. Although sequencing platforms continue to improve, the method and assembly pipeline chosen still influence the final plasmid assembly sequence. Furthermore, few dedicated tools exist for plasmid assembly, especially for de novo assembly. Here, we evaluated short-read, long-read, and hybrid (both short and long reads) de novo assembly pipelines across three replicates of a 24-plasmid library. Consistent with previous characterizations of each sequencing technology, short-read assemblies had issues resolving GC-rich regions, and long-read assemblies commonly had small insertions and deletions, especially in repetitive regions. The hybrid approach facilitated the most accurate, consistent assembly generation and identified mutations relative to the reference sequence. Although Sanger sequencing can be used to verify specific regions, some GC-rich and repetitive regions were difficult to resolve using any method, suggesting that easily sequenced genetic parts should be prioritized in the design of new genetic constructs.
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- Award ID(s):
- 2123367
- PAR ID:
- 10560000
- Publisher / Repository:
- ACSPublications
- Date Published:
- Journal Name:
- ACS Synthetic Biology
- ISSN:
- 2161-5063
- Subject(s) / Keyword(s):
- plasmids, synthetic biology, biotechnology, DNA sequencing, reproducibility, bioinformatics
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract Background Modern sequencing technologies should make the assembly of the relatively small mitochondrial genomes an easy undertaking. However, few tools exist that address mitochondrial assembly directly. Results As part of the Vertebrate Genomes Project (VGP) we develop mitoVGP, a fully automated pipeline for similarity-based identification of mitochondrial reads and de novo assembly of mitochondrial genomes that incorporates both long (> 10 kbp, PacBio or Nanopore) and short (100–300 bp, Illumina) reads. Our pipeline leads to successful complete mitogenome assemblies of 100 vertebrate species of the VGP. We observe that tissue type and library size selection have considerable impact on mitogenome sequencing and assembly. Comparing our assemblies to purportedly complete reference mitogenomes based on short-read sequencing, we identify errors, missing sequences, and incomplete genes in those references, particularly in repetitive regions. Our assemblies also identify novel gene region duplications. The presence of repeats and duplications in over half of the species herein assembled indicates that their occurrence is a principle of mitochondrial structure rather than an exception, shedding new light on mitochondrial genome evolution and organization. Conclusions Our results indicate that even in the “simple” case of vertebrate mitogenomes the completeness of many currently available reference sequences can be further improved, and caution should be exercised before claiming the complete assembly of a mitogenome, particularly from short reads alone.more » « less
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Abstract SummaryPlasCAT (Plasmid Cloud Assembly Tool) is an easy-to-use cloud-based bioinformatics tool that enables de novo plasmid sequence assembly from raw sequencing data. Nontechnical users can now assemble sequences from long reads and short reads without ever touching a line of code. PlasCAT uses high-performance computing servers to reduce run times on assemblies and deliver results faster. Availability and implementationPlasCAT is freely available on the web at https://sequencing.genofab.com. The assembly pipeline source code and server code are available for download at https://bitbucket.org/genofabinc/workspace/projects/PLASCAT. Click the Cancel button to access the source code without authenticating. Web servers implemented in React.js and Python, with all major browsers supported.more » « less
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Abstract Long-read sequencing technology enables significant progress in de novo genome assembly. However, the high error rate and the wide error distribution of raw reads result in a large number of errors in the assembly. Polishing is a procedure to fix errors in the draft assembly and improve the reliability of genomic analysis. However, existing methods treat all the regions of the assembly equally while there are fundamental differences between the error distributions of these regions. How to achieve very high accuracy in genome assembly is still a challenging problem. Motivated by the uneven errors in different regions of the assembly, we propose a novel polishing workflow named BlockPolish. In this method, we divide contigs into blocks with low complexity and high complexity according to statistics of aligned nucleotide bases. Multiple sequence alignment is applied to realign raw reads in complex blocks and optimize the alignment result. Due to the different distributions of error rates in trivial and complex blocks, two multitask bidirectional Long short-term memory (LSTM) networks are proposed to predict the consensus sequences. In the whole-genome assemblies of NA12878 assembled by Wtdbg2 and Flye using Nanopore data, BlockPolish has a higher polishing accuracy than other state-of-the-arts including Racon, Medaka and MarginPolish & HELEN. In all assemblies, errors are predominantly indels and BlockPolish has a good performance in correcting them. In addition to the Nanopore assemblies, we further demonstrate that BlockPolish can also reduce the errors in the PacBio assemblies. The source code of BlockPolish is freely available on Github (https://github.com/huangnengCSU/BlockPolish).more » « less
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Slotte, Tanja (Ed.)Abstract Intracellular transfers of mitochondrial DNA continue to shape nuclear genomes. Chromosome 2 of the model plant Arabidopsis thaliana contains one of the largest known nuclear insertions of mitochondrial DNA (numts). Estimated at over 600 kb in size, this numt is larger than the entire Arabidopsis mitochondrial genome. The primary Arabidopsis nuclear reference genome contains less than half of the numt because of its structural complexity and repetitiveness. Recent data sets generated with improved long-read sequencing technologies (PacBio HiFi) provide an opportunity to finally determine the accurate sequence and structure of this numt. We performed a de novo assembly using sequencing data from recent initiatives to span the Arabidopsis centromeres, producing a gap-free sequence of the Chromosome 2 numt, which is 641 kb in length and has 99.933% nucleotide sequence identity with the actual mitochondrial genome. The numt assembly is consistent with the repetitive structure previously predicted from fiber-based fluorescent in situ hybridization. Nanopore sequencing data indicate that the numt has high levels of cytosine methylation, helping to explain its biased spectrum of nucleotide sequence divergence and supporting previous inferences that it is transcriptionally inactive. The original numt insertion appears to have involved multiple mitochondrial DNA copies with alternative structures that subsequently underwent an additional duplication event within the nuclear genome. This work provides insights into numt evolution, addresses one of the last unresolved regions of the Arabidopsis reference genome, and represents a resource for distinguishing between highly similar numt and mitochondrial sequences in studies of transcription, epigenetic modifications, and de novo mutations.more » « less
- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1021/acssynbio.4c00539
