Dataset Information

Impact of short-read sequencing on the misassembly of a plant genome.

ABSTRACT:

Background

Availability of plant genome sequences has led to significant advances. However, with few exceptions, the great majority of existing genome assemblies are derived from short read sequencing technologies with highly uneven read coverages indicative of sequencing and assembly issues that could significantly impact any downstream analysis of plant genomes. In tomato for example, 0.6% (5.1?Mb) and 9.7% (79.6?Mb) of short-read based assembly had significantly higher and lower coverage compared to background, respectively.

Results

To understand what the causes may be for such uneven coverage, we first established machine learning models capable of predicting genomic regions with variable coverages and found that high coverage regions tend to have higher simple sequence repeat and tandem gene densities compared to background regions. To determine if the high coverage regions were misassembled, we examined a recently available tomato long-read based assembly and found that 27.8% (1.41?Mb) of high coverage regions were potentially misassembled of duplicate sequences, compared to 1.4% in background regions. In addition, using a predictive model that can distinguish correctly and incorrectly assembled high coverage regions, we found that misassembled, high coverage regions tend to be flanked by simple sequence repeats, pseudogenes, and transposon elements.

Conclusions

Our study provides insights on the causes of variable coverage regions and a quantitative assessment of factors contributing to plant genome misassembly when using short reads and the generality of these causes and factors should be tested further in other species.

SUBMITTER: Wang P

PROVIDER: S-EPMC7852129 | biostudies-literature | 2021 Feb

REPOSITORIES: biostudies-literature

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Publications

Impact of short-read sequencing on the misassembly of a plant genome.

Wang Peipei P Meng Fanrui F Moore Bethany M BM Shiu Shin-Han SH

BMC genomics 20210202 1

<h4>Background</h4>Availability of plant genome sequences has led to significant advances. However, with few exceptions, the great majority of existing genome assemblies are derived from short read sequencing technologies with highly uneven read coverages indicative of sequencing and assembly issues that could significantly impact any downstream analysis of plant genomes. In tomato for example, 0.6% (5.1 Mb) and 9.7% (79.6 Mb) of short-read based assembly had significantly higher and lower cover ...[more]

PMID: 33530937

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Project description:MotivationShort-read whole-genome sequencing (WGS) is a vital tool for clinical applications and basic research. Genetic divergence from the reference genome, repetitive sequences and sequencing bias reduces the performance of variant calling using short-read alignment, but the loss in recall and specificity has not been adequately characterized. To benchmark short-read variant calling, we used 36 diverse clinical Mycobacterium tuberculosis (Mtb) isolates dually sequenced with Illumina short-reads and PacBio long-reads. We systematically studied the short-read variant calling accuracy and the influence of sequence uniqueness, reference bias and GC content.ResultsReference-based Illumina variant calling demonstrated a maximum recall of 89.0% and minimum precision of 98.5% across parameters evaluated. The approach that maximized variant recall while still maintaining high precision (<99%) was tuning the mapping quality filtering threshold, i.e. confidence of the read mapping (recall = 85.8%, precision = 99.1%, MQ ≥ 40). Additional masking of repetitive sequence content is an alternative conservative approach to variant calling that increases precision at cost to recall (recall = 70.2%, precision = 99.6%, MQ ≥ 40). Of the genomic positions typically excluded for Mtb, 68% are accurately called using Illumina WGS including 52/168 PE/PPE genes (34.5%). From these results, we present a refined list of low confidence regions across the Mtb genome, which we found to frequently overlap with regions with structural variation, low sequence uniqueness and low sequencing coverage. Our benchmarking results have broad implications for the use of WGS in the study of Mtb biology, inference of transmission in public health surveillance systems and more generally for WGS applications in other organisms.Availability and implementationAll relevant code is available at https://github.com/farhat-lab/mtb-illumina-wgs-evaluation.Supplementary informationSupplementary data are available at Bioinformatics online.

Dataset Information

Impact of short-read sequencing on the misassembly of a plant genome.

Background

Results

Conclusions

Publications

Impact of short-read sequencing on the misassembly of a plant genome.

Similar Datasets

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