Abstract
Unmapped next-generation sequencing reads are typically ignored while they contain biologically relevant information. We systematically analyzed unmapped reads from whole genome sequencing of 33 inbred rat strains. High quality reads were selected and enriched for biologically relevant sequences; similarity-based analysis revealed clustering similar to previously reported phylogenetic trees. Our results demonstrate that on average 20% of all unmapped reads harbor sequences that can be used to improve reference genomes and generate hypotheses on potential genotype-phenotype relationships. Analysis pipelines would benefit from incorporating the described methods and reference genomes would benefit from inclusion of the genomic segments obtained through these efforts.
Highlights
Next-generation sequencing (NGS) is used in a large variety of applications ranging from single cell analyses to complex microbial communities and complete vertebrate and plant genome analyses [1]
We aligned whole genome sequencing (WGS) data of 33 rat strains to the latest rat reference genome assembly (BN/NHsdMcWi, RGSC5.0) to identify ‘unmappable’ reads (Table 1)
By comparing the current rat reference genome with WGS data obtained from the same animal that was used for creating this reference, we found that 39% of the total unmapped reads are due to missing sequences in the reference genome
Summary
Next-generation sequencing (NGS) is used in a large variety of applications ranging from single cell analyses to complex microbial communities and complete vertebrate and plant genome analyses [1]. NGS reads are, in general, aligned to an organism-specific reference genome as a first step in data analysis. Such reference genomes are typically derived from a single individual, animal or strain, with the exception of the human reference genome. Reads that align (map) to the reference genome are subsequently used for data analysis, while the unmapped reads are usually discarded [2,3]. Filtering out reads originating from the first source is fairly straightforward and implemented in most data processing procedures by discarding reads with low quality scores [4,5]. The second source of unmapped reads often contains sequences from exogenous species due to experimental and sampling
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