Fig. 8

Many camouflaged regions can be rescued, including CR1, even in standard short-read sequencing data. Many large-scale whole-genome or whole-exome sequencing projects exist, covering tens of thousands of individuals. All of these datasets are affected by dark and camouflaged regions that may harbor mutations that either drive or modify disease in patients. Ideally, all samples would be re-sequenced using the latest technologies over time, but financial resources and biological samples are limited, making it essential to maximize the utility of existing data. We developed a method to rescue mutations in most camouflaged regions, including for standard short-read sequencing data. When confronted with a sequencing read that aligns to two or more regions equally well (with high confidence), most aligners (e.g., BWA [11,12,13]) will randomly assign the read to one of the regions with a low mapping quality (e.g., MAPQ = 0 for BWA). a Because the reads are already aligned to one of the regions, we can use the following steps to rescue mutations in most camouflaged regions: (1) extract reads from camouflaged regions, (2) mask all highly similar regions in the reference genome, except one, and re-align the extracted reads, (3) call mutations using standard methods (adjusting for ploidy), and (4) determine precise location using targeted sequencing (e.g., long-range PCR combined with Sanger, or targeted long-read sequencing [1]). Without competing camouflaged regions to confuse the aligner, the aligner will assign a high mapping quality, allowing variant callers to behave normally. b Exons 10, 18, and 26 in CR1 are identical, according to the reference genome. Standard aligners will randomly scatter reads matching that sequence across these exons and assign a low mapping quality (e.g., MAPQ = 0 for BWA; indicated as hollow reads). Red lines indicate an individual’s mutation that exists in one of these exons, but reads containing this mutation also get scattered and assigned a low mapping quality. c By masking exons 18 and 26, we can align all of these reads to exon 10 with high mapping qualities to determine whether a mutation exists. We cannot determine at this stage which of the three exons the mutation is actually located in, but researchers can test association with a given disease to determine whether the mutation is worth further investigation. d As a proof of principle, we rescued approximately 4214 exonic variants in the ADSP (TiTv = 2.26) using our method, including a frameshift mutation in CR1 (MAF = 0.00019) that is found in five cases and zero controls (three representative samples shown). The frameshift results in a stop codon shortly downstream. The ADSP is not large enough to formally assess association between the CR1 frameshift and Alzheimer’s disease, but we believe the mutation merits follow-up studies given its location (CR1 binding domain) and CR1’s strong association with disease