Fig. 1

The genome analysis pipeline. Note that, for clarity, some steps have been omitted. Figure illustrations are not to scale and are only meant to be illustrative of the differences between short- and long-read sequencing. a Unaligned reads from sequencing machines are stored as FASTQ file formats. This is a text-based format for storing both a DNA sequence and its corresponding quality scores. b Reads are aligned to the genome. Short reads provide deep coverage, whereas reads that have been sequenced from both ends (blue arrows) help to orientate unaligned contigs. It is difficult to align short reads confidently across repetitive sequences when the repeating genome sequence is longer than the sequence read. Long-read sequences help to order contigs across larger regions, particularly with repetitive sequences, but do not provide the necessary depth needed to be confident of calling a base at a certain position. Note that there is a large region where there is no read coverage at all. This is indicative of structural variation. Here, the patient has a large deletion with respect to the reference genome. Once the reads have been aligned to the reference genome they are stored in a BAM file. A BAM file (.bam) is the binary version of a sequence alignment map (SAM file format). The latter is a tab-delimited text-based format for storing DNA sequences aligned to a reference sequence. c The Variant Call Format (VCF) specifies the format of a text file used in bioinformatics for storing genetic sequence variations. VCF files are much smaller than FASTQ and BAM files. Note that single-nucleotide variants (SNVs) and small insertions and deletions (‘indels’) are illustrated as red and purple blocks, whereas a much larger structural variant is indicated by an orange block