pBACode: a random-barcode-based high-throughput approach for BAC paired-end sequencing and physical clone mapping

Abstract

Applications that use Bacterial Artificial Chromosome (BAC) libraries often require paired-end sequences and knowledge of the physical location of each clone in plates. To facilitate obtaining this information in high-throughput, we generated pBACode vectors: a pool of BAC cloning vectors, each with a pair of random barcodes flanking its cloning site. In a pBACode BAC library, the BAC ends and their linked barcodes can be sequenced in bulk. Barcode pairs are determined by sequencing the empty pBACode vectors, which allows BAC ends to be paired according to their barcodes. For physical clone mapping, the barcodes are used as unique markers for their linked genomic sequence. After multi-dimensional pooling of BAC clones, the barcodes are sequenced and deconvoluted to locate each clone. We generated a pBACode library of 94,464 clones for the flounder Paralichthys olivaceus and obtained paired-end sequence from 95.4% of the clones. Incorporating BAC paired-ends into the genome preassembly improved its continuity by over 10-fold. Furthermore, we were able to use the barcodes to map the physical locations of each clone in just 50 pools, with up to 11 808 clones per pool. Our physical clone mapping located 90.2% of BAC clones, enabling targeted characterization of chromosomal rearrangements.

High-throughput long paired-end sequencing of a Fosmid library by PacBio

“…3). It is almost the same as the previously reported 96% of Fosill [51] and better than the 90.2% of pBACode [52]. Chimaeras were the main factor affecting this parameter and are usually an obstacle in the application of paired-end technology, which could result in misassemblies.…”

“…Their large spans help to resolve long repeats and segmental duplications and provide long-range connectivity to shotgun assemblies of complex genomes [22, 49, 50]. Several high-throughput paired-end sequencing approaches using large-insert genomic libraries, such as the Fosmid library called Fosill (Fosmid libraries by Illumina) [51] and the BAC library called pBACode [52], were developed and used for the de novo assembly and SV detection of several genomes [52, 53]. Also, large insert size paired-ends methods that do not depend on large-insert genomic libraries have been created for large and complex genomes, especially those rich in repeats, such as 10× Genomics [38], Hi-C [54], and BioNano [11, 46].…”

“…However, this method depends on the delicate concentration of DNA polymerase I and dNTPs and has a limit in generating long paired ends. In pBACode method, a random-barcode-based high-throughput approach with ultrasonic interruption was used for BAC paired-end sequencing [52]. This approach can generate single ends of up to 800 bp long and pair them with the same barcode.…”

Genomic basis of recombination suppression in the hybrid between Caenorhabditis briggsae and C. nigoni

“…The clones were inoculated into 96-well plates with LB broth and 12.5 μg/ml chloramphenicol, cultured and then stored in –80°C. Construction of BAC genomic library was outsourced to Genome Resource Laboratory of Huazhong Agricultural University using the methods described previously ( 17 ).…”

“…The PCR products were sequenced on MiSeq at 2 × 250 bp mode or Nanopore sequencing and the end sequences generated were used in the subsequent analysis. Mate-pair sequencing of BAC ends and its data analysis were performed as described previously ( 17 ).…”