Although plastid genome (plastome) structure is highly conserved across most seed plants, investigations during the past two decades have revealed several disparately related lineages that experienced substantial rearrangements. Most plastomes contain a large inverted repeat and two single‐copy regions, and a few dispersed repeats; however, the plastomes of some taxa harbour long repeat sequences (>300 bp). These long repeats make it challenging to assemble complete plastomes using short‐read data, leading to misassemblies and consensus sequences with spurious rearrangements. Single‐molecule, long‐read sequencing has the potential to overcome these challenges, yet there is no consensus on the most effective method for accurately assembling plastomes using long‐read data. We generated a pipeline, plastid Genome Assembly Using Long‐read data (ptGAUL), to address the problem of plastome assembly using long‐read data from Oxford Nanopore Technologies (ONT) or Pacific Biosciences platforms. We demonstrated the efficacy of the ptGAUL pipeline using 16 published long‐read data sets. We showed that ptGAUL quickly produces accurate and unbiased assemblies using only ~50× coverage of plastome data. Additionally, we deployed ptGAUL to assemble four new Juncus (Juncaceae) plastomes using ONT long reads. Our results revealed many long repeats and rearrangements in Juncus plastomes compared with basal lineages of Poales. The ptGAUL pipeline is available on GitHub: https://github.com/Bean061/ptgaul.

Although plastid genome (plastome) structure is highly conserved across most seed plants, investigations during the past two decades have revealed several disparately related lineages that have experienced substantial rearrangements. Most plastomes have two inverted repeat regions and two single-copy regions with few dispersed repeats. However, the plastomes of some taxa do harbor long repeat sequences (>300 bp). These long repeats make it difficult to assemble complete plastomes using short read data, leading to misassemblies and consensus sequences that have spurious rearrangements. Long read sequencing can potentially overcome these challenges. However, there is no consensus as to the most effective method for accurately assembling plastomes using long read data. Here, we generated a pipeline, plastid Genome Assembly Using Long-read data (ptGAUL) to address the problem of assembling of plastomes using long read data from Oxford Nanopore Technologies (ONT) or Pacific Biosciences (Pacbio) platforms. We demonstrated the efficacy of the ptGAUL pipeline using 16 published long read datasets. We showed that ptGAUL produces accurate and unbiased assemblies. Additionally, we applied ptGAUL to assemble four Juncus (Juncaceae) plastomes using ONT long reads. Our results revealed many long repeats and rearrangements in Juncus plastomes compared with basal lineages of Poales.

Although plastid genome (plastome) structure is highly conserved across most seed plants, investigations during the past two decades revealed several disparately related lineages that experienced substantial rearrangements. Most plastomes contain a large, inverted repeat and two single-copy regions and few dispersed repeats, however the plastomes of some taxa harbor long repeat sequences (>300 bp). These long repeats make it difficult to assemble complete plastomes using short-read data leading to misassemblies and consensus sequences that have spurious rearrangements. Single-molecule, long-read sequencing has the potential to overcome these challenges, yet there is no consensus on the most effective method for accurately assembling plastomes using long-read data. We generated a pipeline, plastid Genome Assembly Using Long-read data (ptGAUL), to address the problem of plastome assembly using long-read data from Oxford Nanopore Technologies (ONT) or Pacific Biosciences platforms. We demonstrated the efficacy of the ptGAUL pipeline using 16 published long-read datasets. We showed that ptGAUL produces accurate and unbiased assemblies. Additionally, we employed ptGAUL to assemble four new Juncus (Juncaceae) plastomes using ONT long reads. Our results revealed many long repeats and rearrangements in Juncus plastomes compared with basal lineages of Poales.