Abstract
BACKGROUND: Many medicinal plants are known for their complex genomes with high ploidy, heterozygosity, and repetitive content which pose severe challenges for genome sequencing of those species. Long reads from Oxford nanopore sequencing technology (ONT) or Pacific Biosciences Single Molecule, Real-Time (SMRT) sequencing offer great advantages in de novo genome assembly, especially for complex genomes with high heterozygosity and repetitive content. Currently, multiple allotetraploid species have sequenced their genomes by long-read sequencing. However, we found that a considerable proportion of these genomes (7.9% on average, maximum 23.7%) could not be covered by NGS (Next Generation Sequencing) reads (uncovered region by NGS reads, UCR) suggesting the questionable and low-quality of those area or genomic areas that can't be sequenced by NGS due to sequencing bias. The underlying causes of those UCR in the genome assembly and solutions to this problem have never been studied. METHODS: In the study, we sequenced the tetraploid genome of Veratrum dahuricum (Turcz.) O. Loes (VDL), a Chinese medicinal plant, with ONT platform and assembled the genome with three strategies in parallel. We compared the qualities, coverage, and heterozygosity of the three ONT assemblies with another released assembly of the same individual using reads from PacBio circular consensus sequencing (CCS) technology, to explore the cause of the UCR. RESULTS: By mapping the NGS reads against the three ONT assemblies and the CCS assembly, we found that the coverage of those ONT assemblies by NGS reads ranged from 49.15 to 76.31%, much smaller than that of the CCS assembly (99.53%). And alignment between ONT assemblies and CCS assembly showed that most UCR can be aligned with CCS assembly. So, we conclude that the UCRs in ONT assembly are low-quality sequences with a high error rate that can't be aligned with short reads, rather than genomic regions that can't be sequenced by NGS. Further comparison among the intermediate versions of ONT assemblies showed that the most probable origin of those errors is a combination of artificial errors introduced by "self-correction" and initial sequencing error in long reads. We also found that polishing the ONT assembly with CCS reads can correct those errors efficiently. CONCLUSIONS: Through analyzing genome features and reads alignment, we have found the causes for the high proportion of UCR in ONT assembly of VDL are sequencing errors and additional errors introduced by self-correction. The high error rates of ONT-raw reads make them not suitable for self-correction prior to allotetraploid genome assembly, as the self-correction will introduce artificial errors to > 5% of the UCR sequences. We suggest high-precision CCS reads be used to polish the assembly to correct those errors effectively for polyploid genomes.
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Claims 9
Claim · model-written summary
The coverage of the three ONT assemblies of Veratrum dahuricum by NGS reads ranged from 49.15 to 76.31%, much smaller than that of the CCS assembly.
- Taxon
- Veratrum dahuricum
Veratrum dahuricume0 - Outcome
- coverage of those ONT assemblies by NGS readse1
- Value
- 49.15 to 76.31%e1
- Comparator
- CCS assemblye1
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“Veratrum dahuricum”
e1“the coverage of those ONT assemblies by NGS reads ranged from 49.15 to 76.31%, much smaller than that of the CCS assembly (99.53%)”
Claim · model-written summary
The CCS assembly of Veratrum dahuricum was covered by NGS reads at 99.53%, far above the ONT assemblies.
- Taxon
- Veratrum dahuricumVeratrum dahuricume0
- Outcome
- coveragee1
- Value
- 99.53%e1
- Comparator
- ONT assembliese1
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“Veratrum dahuricum”
e1“the coverage of those ONT assemblies by NGS reads ranged from 49.15 to 76.31%, much smaller than that of the CCS assembly (99.53%)”
Claim · model-written summary
Alignment between ONT assemblies and the CCS assembly showed that most uncovered regions (UCR) could be aligned with the CCS assembly.
- Taxon
- Veratrum dahuricumVeratrum dahuricume0
- Outcome
- UCRe1
- Comparator
- CCS assemblye1
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“Veratrum dahuricum”
e1“most UCR can be aligned with CCS assembly”
Claim · model-written summary
The uncovered regions in the ONT assembly are low-quality sequences with a high error rate that cannot be aligned with short reads, not unsequenceable genomic regions.
- Taxon
- Veratrum dahuricumVeratrum dahuricume0
- Outcome
- low-quality sequences with a high error ratee1
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“Veratrum dahuricum”
e1“the UCRs in ONT assembly are low-quality sequences with a high error rate that can't be aligned with short reads”
Claim · model-written summary
The most probable origin of errors in the ONT assemblies is a combination of artificial errors introduced by self-correction and initial sequencing error in long reads.
- Taxon
- Veratrum dahuricumVeratrum dahuricume0
- Treatment
- self-correctione1
- Outcome
- errorse1
- Qualifier
- initial sequencing error in long readse1
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“Veratrum dahuricum”
e1“the most probable origin of those errors is a combination of artificial errors introduced by "self-correction" and initial sequencing error in long reads”
Claim · model-written summary
Polishing the ONT assembly with CCS reads corrected the errors efficiently.
- Taxon
- Veratrum dahuricumVeratrum dahuricume0
- Treatment
- polishing the ONT assembly with CCS readse1
- Outcome
- errorse1
- Direction
- correcte1
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“Veratrum dahuricum”
e1“polishing the ONT assembly with CCS reads can correct those errors efficiently”
Claim · model-written summary
Self-correction of ONT reads introduces artificial errors into more than 5% of the UCR sequences.
- Taxon
- Veratrum dahuricumVeratrum dahuricume0
- Treatment
- self-correctione1
- Outcome
- artificial errorse1
- Direction
- introducee1
- Value
- > 5%e1
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“Veratrum dahuricum”
e1“the self-correction will introduce artificial errors to > 5% of the UCR sequences”
Claim · model-written summary
The authors suggest using high-precision CCS reads to polish the assembly to correct errors effectively for polyploid genomes.
- Taxon
- Veratrum dahuricumVeratrum dahuricume0
- Outcome
- polish the assembly to correct those errorse1
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“Veratrum dahuricum”
e1“We suggest high-precision CCS reads be used to polish the assembly to correct those errors effectively for polyploid genomes”
Claim · model-written summary
A considerable proportion of published allotetraploid long-read genomes (7.9% on average, maximum 23.7%) could not be covered by NGS reads.
Verbatim
e0“Currently, multiple allotetraploid species have sequenced their genomes by long-read sequencing”
e1“we found that a considerable proportion of these genomes (7.9% on average, maximum 23.7%) could not be covered by NGS (Next Generation Sequencing) reads”