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Impact of index hopping and bias towards the reference allele on accuracy of genotype calls from low-coverage sequencing.

ABSTRACT: BACKGROUND:Inherent sources of error and bias that affect the quality of sequence data include index hopping and bias towards the reference allele. The impact of these artefacts is likely greater for low-coverage data than for high-coverage data because low-coverage data has scant information and many standard tools for processing sequence data were designed for high-coverage data. With the proliferation of cost-effective low-coverage sequencing, there is a need to understand the impact of these errors and bias on resulting genotype calls from low-coverage sequencing. RESULTS:We used a dataset of 26 pigs sequenced both at 2× with multiplexing and at 30× without multiplexing to show that index hopping and bias towards the reference allele due to alignment had little impact on genotype calls. However, pruning of alternative haplotypes supported by a number of reads below a predefined threshold, which is a default and desired step of some variant callers for removing potential sequencing errors in high-coverage data, introduced an unexpected bias towards the reference allele when applied to low-coverage sequence data. This bias reduced best-guess genotype concordance of low-coverage sequence data by 19.0 absolute percentage points. CONCLUSIONS:We propose a simple pipeline to correct the preferential bias towards the reference allele that can occur during variant discovery and we recommend that users of low-coverage sequence data be wary of unexpected biases that may be produced by bioinformatic tools that were designed for high-coverage sequence data.

SUBMITTER: Ros-Freixedes R

PROVIDER: S-EPMC6293637 | biostudies-literature | 2018 Dec

REPOSITORIES: biostudies-literature

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Impact of index hopping and bias towards the reference allele on accuracy of genotype calls from low-coverage sequencing.

Ros-Freixedes Roger R Battagin Mara M Johnsson Martin M Gorjanc Gregor G Mileham Alan J AJ Rounsley Steve D SD Hickey John M JM

Genetics, selection, evolution : GSE 20181213 1

<h4>Background</h4>Inherent sources of error and bias that affect the quality of sequence data include index hopping and bias towards the reference allele. The impact of these artefacts is likely greater for low-coverage data than for high-coverage data because low-coverage data has scant information and many standard tools for processing sequence data were designed for high-coverage data. With the proliferation of cost-effective low-coverage sequencing, there is a need to understand the impact ...[more]

PMID: 30545283

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Project description:MotivationGenome-wide association studies (GWAS) are used to discover genes underlying complex, heritable disorders for which less powerful study designs have failed in the past. The number of GWAS has skyrocketed recently with findings reported in top journals and the mainstream media. Microarrays are the genotype calling technology of choice in GWAS as they permit exploration of more than a million single nucleotide polymorphisms (SNPs) simultaneously. The starting point for the statistical analyses used by GWAS to determine association between loci and disease is making genotype calls (AA, AB or BB). However, the raw data, microarray probe intensities, are heavily processed before arriving at these calls. Various sophisticated statistical procedures have been proposed for transforming raw data into genotype calls. We find that variability in microarray output quality across different SNPs, different arrays and different sample batches have substantial influence on the accuracy of genotype calls made by existing algorithms. Failure to account for these sources of variability can adversely affect the quality of findings reported by the GWAS.ResultsWe developed a method based on an enhanced version of the multi-level model used by CRLMM version 1. Two key differences are that we now account for variability across batches and improve the call-specific assessment of each call. The new model permits the development of quality metrics for SNPs, samples and batches of samples. Using three independent datasets, we demonstrate that the CRLMM version 2 outperforms CRLMM version 1 and the algorithm provided by Affymetrix, Birdseed. The main advantage of the new approach is that it enables the identification of low-quality SNPs, samples and batches.AvailabilitySoftware implementing of the method described in this article is available as free and open source code in the crlmm R/BioConductor package.Supplementary informationSupplementary data are available at Bioinformatics online.

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Project description:Natural ecological variability and analytical design can bias the derived value of a biotic index through the variable influence of indicator body-size, abundance, richness, and ascribed tolerance scores. Descriptive statistics highlight this risk for 26 aquatic indicator systems; detailed analysis is provided for contrasting weighted-average indices applying the example of the BMWP, which has the best supporting data. Differences in body size between taxa from respective tolerance classes is a common feature of indicator systems; in some it represents a trend ranging from comparatively small pollution tolerant to larger intolerant organisms. Under this scenario, the propensity to collect a greater proportion of smaller organisms is associated with negative bias however, positive bias may occur when equipment (e.g. mesh-size) selectively samples larger organisms. Biotic indices are often derived from systems where indicator taxa are unevenly distributed along the gradient of tolerance classes. Such skews in indicator richness can distort index values in the direction of taxonomically rich indicator classes with the subsequent degree of bias related to the treatment of abundance data. The misclassification of indicator taxa causes bias that varies with the magnitude of the misclassification, the relative abundance of misclassified taxa and the treatment of abundance data. These artifacts of assessment design can compromise the ability to monitor biological quality. The statistical treatment of abundance data and the manipulation of indicator assignment and class richness can be used to improve index accuracy. While advances in methods of data collection (i.e. DNA barcoding) may facilitate improvement, the scope to reduce systematic bias is ultimately limited to a strategy of optimal compromise. The shortfall in accuracy must be addressed by statistical pragmatism. At any particular site, the net bias is a probabilistic function of the sample data, resulting in an error variance around an average deviation. Following standardized protocols and assigning precise reference conditions, the error variance of their comparative ratio (test-site:reference) can be measured and used to estimate the accuracy of the resultant assessment.

Dataset Information

Impact of index hopping and bias towards the reference allele on accuracy of genotype calls from low-coverage sequencing.

Publications

Impact of index hopping and bias towards the reference allele on accuracy of genotype calls from low-coverage sequencing.

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