Long reads are also less susceptible to issues caused by GC rich, complex, repetitive, or heterozygous regions as well as structural variations in the DNA 19, 20, 21, 22. This is particularly important when the DNA sample contains similar sequences, as the assembly of short reads can lead to chimeric contigs. Such long-read sequencing techniques are starting to gain traction because they remove the gene assembly process, or polishing, necessary when handling short reads 3. Furthermore, emerging third generation sequencing technologies, such as PacBio and Oxford Nanopore, can sequence much longer reads 17, 18. Notable achievements include the ability to investigate local fitness landscapes of proteins or nucleic acids, to perform in-depth structure to function studies, and to analyse local and global regions of proteins 12, 13, 14, 15.Īt the forefront of NGS technologies are Illumina sequencing methods, which can sequence 150–600 base pairs (bp) and up to one billion sequences 16. However, NGS technologies have enabled simultaneous high-throughput sequencing of millions to billions of DNA molecules and allowed for a more comprehensive analysis of selection and screening outputs, thereby probing a greater proportion of the potential sequence space 10, 11. Such studies face challenges resulting from the limitations of DNA-sequencing technologies, in particular when reliant on laborious and expensive Sanger sequencing-based methods 8, 9. In molecular engineering, large mutational libraries of DNA encoding protein variants are frequently used to select, screen, or probe for specific properties or activities 6, 7.
Consequently, advances in DNA sequencing technology bring benefits to many fields of research, including molecular engineering 5. Next generation sequencing (NGS) approaches have improved our ability to study genomes and large libraries of DNA for a fraction of previous costs 2, 3, 4. Over the past decade, advances in DNA sequencing technology have accelerated at an unprecedented rate 1. The improved method is accompanied by a simple and user-friendly analysis pipeline, DeCatCounter, to sequence medium-length sequences efficiently at one-fifth of the cost. We applied this efficient concatenation protocol to sequence nine DNA populations from a protein engineering study. Our method improved upon previously published concatenation attempts, leading to a greater sequencing depth, high-quality reads and limited sample preparation at little expense. We optimised a simple, robust method to concatenate genes of ~ 870 bp five times and then sequenced the resulting DNA of ~ 5,000 bp by PacBioSMRT long-read sequencing. We sought to sequence several DNA populations of ~ 870 bp in length with a sequencing accuracy of 99% and to the greatest depth possible. For example, the PacBio Sequel I system yields ~ 100,000–300,000 reads with an accuracy per base pair of 90–99%. Methods for sequencing mid-sized sequences of 600–5,000 bp are currently less efficient.
Main developments have focused on either sequencing many short sequences or fewer large sequences. Advances in sequencing technology have allowed researchers to sequence DNA with greater ease and at decreasing costs.
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