Data from: Batch effects in a multi-year sequencing study: false biological trends due to changes in read lengths

High-throughput sequencing is a powerful tool, but suffers biases and errors that must be accounted for to prevent false biological conclusions. Such errors include batch effects, technical errors only present in subsets of data due to procedural changes within a study. If overlooked and multiple batches of data are combined, spurious biological signals can arise, particularly if batches of data are correlated with biological variables. Batch effects can be minimized through randomisation of sample groups across batches. However, in long-term or multi-year studies where data are added incrementally, full randomisation is impossible and batch effects may be a common feature. Here we present a case study where false signals of selection were detected due to a batch effect in a multi-year study of Alpine ibex (Capra ibex). The batch effect arose because sequencing read length changed over the course of the project and populations were added incrementally to the study, resulting in non-random distributions of populations across read lengths. The differences in read length caused small misalignments in a subset of the data, leading to false variant alleles and thus false SNPs. Pronounced allele frequency differences between populations arose at these SNPs because of the correlation between read length and population. This created highly statistically significant, but biologically spurious, signals of selection and false associations between allele frequencies and the environment. We highlight the risk of batch effects and discuss strategies to reduce the impacts of batch effects in multi-year high-throughput sequencing studies.False SNPs caused by batch effectThis file is a vcf file containing the SNP calls at the eight SNPs identified as outliers. Seven of these SNPs were false variants caused by differences in read length.outlier.snps.C.ibex.batch.effects.recode.vcfdemultiplexing.file.Alpine.ibex.Capra.ibexThe demultiplexing file. Includes the sample name, population, sex, the barcodes used, sequencing length and data generator.VCF_filterPolym.Lischer.H.E.LScript to remove SNPs only polymorphic relative to the genome. This was used to remove SNPs when aligning Capra ibex RADseq to the Capra domestica genome