Dataset Information

Assessing reproducibility of inherited variants detected with short-read whole genome sequencing.

ABSTRACT:

Background

Reproducible detection of inherited variants with whole genome sequencing (WGS) is vital for the implementation of precision medicine and is a complicated process in which each step affects variant call quality. Systematically assessing reproducibility of inherited variants with WGS and impact of each step in the process is needed for understanding and improving quality of inherited variants from WGS.

Results

To dissect the impact of factors involved in detection of inherited variants with WGS, we sequence triplicates of eight DNA samples representing two populations on three short-read sequencing platforms using three library kits in six labs and call variants with 56 combinations of aligners and callers. We find that bioinformatics pipelines (callers and aligners) have a larger impact on variant reproducibility than WGS platform or library preparation. Single-nucleotide variants (SNVs), particularly outside difficult-to-map regions, are more reproducible than small insertions and deletions (indels), which are least reproducible when > 5 bp. Increasing sequencing coverage improves indel reproducibility but has limited impact on SNVs above 30×.

Conclusions

Our findings highlight sources of variability in variant detection and the need for improvement of bioinformatics pipelines in the era of precision medicine with WGS.

SUBMITTER: Pan B

PROVIDER: S-EPMC8722114 | biostudies-literature | 2022 Jan

REPOSITORIES: biostudies-literature

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Publications

Assessing reproducibility of inherited variants detected with short-read whole genome sequencing.

Pan Bohu B Ren Luyao L Onuchic Vitor V Guan Meijian M Kusko Rebecca R Bruinsma Steve S Trigg Len L Scherer Andreas A Ning Baitang B Zhang Chaoyang C Glidewell-Kenney Christine C Xiao Chunlin C Donaldson Eric E Sedlazeck Fritz J FJ Schroth Gary G Yavas Gokhan G Grunenwald Haiying H Chen Haodong H Meinholz Heather H Meehan Joe J Wang Jing J Yang Jingcheng J Foox Jonathan J Shang Jun J Miclaus Kelci K Dong Lianhua L Shi Leming L Mohiyuddin Marghoob M Pirooznia Mehdi M Gong Ping P Golshani Rooz R Wolfinger Russ R Lababidi Samir S Sahraeian Sayed Mohammad Ebrahim SME Sherry Steve S Han Tao T Chen Tao T Shi Tieliu T Hou Wanwan W Ge Weigong W Zou Wen W Guo Wenjing W Bao Wenjun W Xiao Wenzhong W Fan Xiaohui X Gondo Yoichi Y Yu Ying Y Zhao Yongmei Y Su Zhenqiang Z Liu Zhichao Z Tong Weida W Xiao Wenming W Zook Justin M JM Zheng Yuanting Y Hong Huixiao H

Genome biology 20220103 1

<h4>Background</h4>Reproducible detection of inherited variants with whole genome sequencing (WGS) is vital for the implementation of precision medicine and is a complicated process in which each step affects variant call quality. Systematically assessing reproducibility of inherited variants with WGS and impact of each step in the process is needed for understanding and improving quality of inherited variants from WGS.<h4>Results</h4>To dissect the impact of factors involved in detection of inh ...[more]

PMID: 34980216

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Project description:The rates of antibiotic resistance of Helicobacter pylori are increasing, and the patterns of resistance are region and population specific. Here, we elucidated the antibiotic resistance pattern of H. pylori in a single center in China and compared short-read- and long-read-based whole-genome sequencing for identifying the genotypes. Resistance rates of 38.5%, 61.5%, 27.9%, and 13.5% against clarithromycin, metronidazole, levofloxacin, and amoxicillin were determined, respectively, while no strain was resistant to tetracycline or furazolidone. Single nucleotide variations (SNVs) in the 23S rRNA and GyrA/B genes revealed by Illumina short-read sequencing showed good diagnostic abilities for clarithromycin and levofloxacin resistance, respectively. Nanopore long-read sequencing also showed a good efficiency in elucidating SNVs in the 23S rRNA gene and, thus, a good ability to detect clarithromycin resistance. The two technologies displayed good consistency in discovering SNVs and shared 76% of SNVs detected in the rRNA gene. Taking Sanger sequencing as the gold standard, Illumina short-read sequencing showed a slightly higher accuracy for discovering SNVs than Nanopore sequencing. There are two copies of the rRNA gene in the genome of H. pylori, and we found that the two copies were not the same in at least 26% of the strains tested, indicating their heterozygous status. Especially, three strains harboring a 2143G/A heterozygous status in the 23S rRNA gene, which is the most important site for clarithromycin resistance, were found. In conclusion, our results provide evidence for an empirical first-line treatment for H. pylori eradication in clinical settings. Moreover, we show that Nanopore sequencing is a potential tool for predicting clarithromycin resistance. IMPORTANCE Helicobacter pylori resistance has been increasing in recent years. The resistance profile, which is important for empirical treatment, is region and population specific. We found high rates of resistance to metronidazole, clarithromycin, and levofloxacin in H. pylori in our center, while no resistance to tetracycline or furazolidone was found. These results provide a reference for local physicians prescribing antibiotics for H. pylori eradication. Nanopore sequencing recently appeared to be a promising technology for elucidating whole-genome sequences, which generates long sequencing reads and is time-efficient and portable. However, a relatively higher error rate of sequencing reads was also found. In this study, we compared Nanopore sequencing and Illumina sequencing for revealing single nucleotide variations in the 23S rRNA gene, which determines clarithromycin resistance, and we found that although there were a few false discoveries, Nanopore sequencing showed good consistency with Illumina sequencing, indicating that it is a potential tool for predicting clarithromycin resistance.

Dataset Information

Assessing reproducibility of inherited variants detected with short-read whole genome sequencing.

Background

Results

Conclusions

Publications

Assessing reproducibility of inherited variants detected with short-read whole genome sequencing.

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