Abstract
BackgroundLarge insert paired-end sequencing technologies are important tools for assembling genomes, delineating associated breakpoints and detecting structural rearrangements. To facilitate the comprehensive detection of inter- and intra-chromosomal structural rearrangements or variants (SVs) and complex genome assembly with long repeats and segmental duplications, we developed a new method based on single-molecule real-time synthesis sequencing technology for generating long paired-end sequences of large insert DNA libraries.ResultsA Fosmid vector, pHZAUFOS3, was developed with the following new features: (1) two 18-bp non-palindromic I-SceI sites flank the cloning site, and another two sites are present in the skeleton of the vector, allowing long DNA inserts (and the long paired-ends in this paper) to be recovered as single fragments and the vector (~ 8 kb) to be fragmented into 2–3 kb fragments by I-SceI digestion and therefore was effectively removed from the long paired-ends (5–10 kb); (2) the chloramphenicol (Cm) resistance gene and replicon (oriV), necessary for colony growth, are located near the two sides of the cloning site, helping to increase the proportion of the paired-end fragments to single-end fragments in the paired-end libraries. Paired-end libraries were constructed by ligating the size-selected, mechanically sheared pooled Fosmid DNA fragments to the Ampicillin (Amp) resistance gene fragment and screening the colonies with Cm and Amp. We tested this method on yeast and Setaria italica Yugu1. Fosmid-size paired-ends with an average length longer than 2 kb for each end were generated. The N50 scaffold lengths of the de novo assemblies of the yeast and S. italica Yugu1 genomes were significantly improved. Five large and five small structural rearrangements or assembly errors spanning tens of bp to tens of kb were identified in S. italica Yugu1 including deletions, inversions, duplications and translocations.ConclusionsWe developed a new method for long paired-end sequencing of large insert libraries, which can efficiently improve the quality of de novo genome assembly and identify large and small structural rearrangements or assembly errors.
Highlights
Large insert paired-end sequencing technologies are important tools for assembling genomes, delineating associated breakpoints and detecting structural rearrangements
The pipeline of high‐throughput long paired‐end sequencing of a Fosmid library To enrich the approaches of genome sequencing, we developed a new method to generate high-throughput long paired-end fragments of a Fosmid library
Size selected DNA fragments were recovered by electroelution, end-repaired and ligated to the Ampicillin resistance gene label
Summary
Large insert paired-end sequencing technologies are important tools for assembling genomes, delineating associated breakpoints and detecting structural rearrangements. Dai et al Plant Methods (2019) 15:142 high throughput, long read length and other advantages, that create a new era of biological sequencing, their disadvantages, such as a high error rate, can not be ignored These DNA sequencing technologies are being rapidly developed and updated, and are widely used in de novo assembly [3, 4], individual genome resequencing [11,12,13,14], clinical applications such as non-invasive prenatal testing [15, 16], and counting devices for a wide range of biochemical or analytical phenomena [1]. Genomic libraries are collections of genomic DNA from a certain species that has been fragmented into specific sizes by biological, chemical or physical disruption They are important tools and materials for molecular cloning, genomic structure and functional characteristic research [17]. Large-insert genomic libraries, such as Fosmid libraries (average insert approximately 40 kb) [18] and BAC library (average insert > 100 kb) [19,20,21], are widely used in physical map construction, genomewide sequencing, comparative genomics research, and genomic resource conservation due to their capacity for long lengths of foreign DNA fragments
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