Complete chloroplast genome of Zingiber mioga by de novo sequencing.
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ABSTRACT:
SUBMITTER: Jiang P
PROVIDER: S-EPMC8008884 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The chloroplast genome of Zingiber striolatum Diels was sequenced using the MGI paired-end sequencing method and assembled. The chloroplast genome was 163,711 bp in length, containing a large single-copy (LSC) region of 88,205 bp, a small single-copy (SSC) region of 15,750 bp, and two inverted repeat (IR) regions of 29,752 bp. The overall GC content was 36.1%, whereas the corresponding value in the IR regions was 41.1%, which was higher than that in the LSC region (33.8%) and SSC region (29.6%). A total of 136 complete genes were annotated in the chloroplast genome of Z. striolatum, including 87 protein-coding genes (79 protein-coding gene species), 40 tRNA genes (29 tRNA species), and 8 rRNA genes (4 rRNA species). A phylogenetic tree was constructed using the maximum likelihood (ML) method, and the results showed that the phylogeny of Zingiber was well resolved with high support values, and Z. striolatum was sister to Z. mioga. The assembly and sequence analysis of the chloroplast genome can provide a basis for developing high-resolution genetic makers.
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Project description:Third generation sequencing methods, like SMRT (Single Molecule, Real-Time) sequencing developed by Pacific Biosciences, offer much longer read length in comparison to Next Generation Sequencing (NGS) methods. Hence, they are well suited for de novo- or re-sequencing projects. Sequences generated for these purposes will not only contain reads originating from the nuclear genome, but also a significant amount of reads originating from the organelles of the target organism. These reads are usually discarded but they can also be used for an assembly of organellar replicons. The long read length supports resolution of repetitive regions and repeats within the organelles genome which might be problematic when just using short read data. Additionally, SMRT sequencing is less influenced by GC rich areas and by long stretches of the same base.We describe a workflow for a de novo assembly of the sugar beet (Beta vulgaris ssp. vulgaris) chloroplast genome sequence only based on data originating from a SMRT sequencing dataset targeted on its nuclear genome. We show that the data obtained from such an experiment are sufficient to create a high quality assembly with a higher reliability than assemblies derived from e.g. Illumina reads only. The chloroplast genome is especially challenging for de novo assembling as it contains two large inverted repeat (IR) regions. We also describe some limitations that still apply even though long reads are used for the assembly.SMRT sequencing reads extracted from a dataset created for nuclear genome (re)sequencing can be used to obtain a high quality de novo assembly of the chloroplast of the sequenced organism. Even with a relatively small overall coverage for the nuclear genome it is possible to collect more than enough reads to generate a high quality assembly that outperforms short read based assemblies. However, even with long reads it is not always possible to clarify the order of elements of a chloroplast genome sequence reliantly which we could demonstrate with Fosmid End Sequences (FES) generated with Sanger technology. Nevertheless, this limitation also applies to short read sequencing data but is reached in this case at a much earlier stage during finishing.
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Project description:BackgroundSecond generation sequencing has permitted detailed sequence characterisation at the whole genome level of a growing number of non-model organisms, but the data produced have short read-lengths and biased genome coverage leading to fragmented genome assemblies. The PacBio RS long-read sequencing platform offers the promise of increased read length and unbiased genome coverage and thus the potential to produce genome sequence data of a finished quality containing fewer gaps and longer contigs. However, these advantages come at a much greater cost per nucleotide and with a perceived increase in error-rate. In this investigation, we evaluated the performance of the PacBio RS sequencing platform through the sequencing and de novo assembly of the Potentilla micrantha chloroplast genome.ResultsFollowing error-correction, a total of 28,638 PacBio RS reads were recovered with a mean read length of 1,902 bp totalling 54,492,250 nucleotides and representing an average depth of coverage of 320× the chloroplast genome. The dataset covered the entire 154,959 bp of the chloroplast genome in a single contig (100% coverage) compared to seven contigs (90.59% coverage) recovered from an Illumina data, and revealed no bias in coverage of GC rich regions. Post-assembly the data were largely concordant with the Illumina data generated and allowed 187 ambiguities in the Illumina data to be resolved. The additional read length also permitted small differences in the two inverted repeat regions to be assigned unambiguously.ConclusionsThis is the first report to our knowledge of a chloroplast genome assembled de novo using PacBio sequence data. The PacBio RS data generated here were assembled into a single large contig spanning the P. micrantha chloroplast genome, with a higher degree of accuracy than an Illumina dataset generated at a much greater depth of coverage, due to longer read lengths and lower GC bias in the data. The results we present suggest PacBio data will be of immense utility for the development of genome sequence assemblies containing fewer unresolved gaps and ambiguities and a significantly smaller number of contigs than could be produced using short-read sequence data alone.
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Project description:Premise:A new set of primers was developed for sequencing of whole chloroplast genomes of Magnolia species and gap-filling of unfinished genomes. Methods and Results:Two hundred and fifty primers were newly designed based on two previously reported chloroplast genomes from two different genera in Magnoliaceae. A total of 134 primer pairs, including the ones developed in this study and 18 previously reported ones, were enough to cover the entire chloroplast genome sequences in Magnoliaceae. Four species from different sections of Magnolia (M. dealbata, M. fraseri var. pyramidata, M. liliiflora, and M. odora) were used to show the general application of these primers to chloroplast genome sequencing in Magnolia. Conclusions:Using the developed primers, four Magnolia chloroplast genomes were successfully assembled. These results show the utility of these primers across Magnolia and their potential use for phylogenetic studies, DNA barcoding, and population genetics in this group.
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