Average Insert Size Research Articles

Genome Sequence Resource of Albifimbria verrucaria Causing the Leaf Spot Disease of the Spinach Plant Spinacia oleracea.

Genome Sequence Resource of Albifimbria verrucaria Causing the Leaf Spot Disease of the Spinach Plant Spinacia oleracea.

Genome Sequence Resource of Sarocladium terricola TR, an Endophytic Fungus as a Potential Biocontrol Agent Against Meloidogyne incognita.

Genome Sequence Resource of Sarocladium terricola TR, an Endophytic Fungus as a Potential Biocontrol Agent Against Meloidogyne incognita.

Exploring the Cunninghamia lanceolata(Lamb.) Hook Genome by BAC Sequencing.

Cunninghamia lanceolata (Lamb.)Hook is an important economic timber tree in China. However, its genome characteristics have not been extensively assessed. To better understand its genome information, the bacterial artificial chromosome (BAC) library of chinese fir was constructed. A total of 422 BAC clones were selected and divided into 10 pools and sequenced, and with an average insert size of 121 kb, ranging from 97 to 145 kb. A total of 61,902,523 bp of reference sequences were sequenced and assembled, and based on an estimated genome size of 11.6 Gb for Chinese fir, the BAC library was estimated to have a total coverage of 0.53% genome equivalents. Bioinformatics analyses were also performed for repeated sequences, tRNAs, coding gene prediction, and functional annotation. The results of this study provide insights into the brief structure of the Chinese fir genome and has generated gene data that will facilitate molecular investigations on the mechanisms underlying tree growth.

Occurrence of Soybean Yellow Common Mosaic Virus in Soybean in China Showing Yellow Common Mosaic Disease.

Soybean yellow common mosaic virus (SYCMV), a positive sense ssRNA virus classified in the genus Sobemovirus, was first reported and characterized in Korea (Nam et al., 2012). Currently, its only known host is soybean (Nam et al., 2012) on which it causes bright yellow mosaic and crinkling of the leaves (Lim et al., 2016). During a field survey in July 2019, bright yellow mosaic and mild crinkling symptoms were observed on soybean leaves (cv. Zhonghuang 13) in the Hubei province of China. To identify the possible pathogen(s) associated to the disease symptoms, leaves from five symptomatic plants were collected, pooled and total RNA was extracted using TRIzol® Reagent (Invitrogen, CA, USA). 10 μg of the total RNA was purified via magnetic beads (Thermo Fischer Scientific, USA) and a TruSeq RNA Sample Prep Kit (Illumina, San Diego, CA, USA) was then used to construct an RNA sequencing library. Transcriptome sequencing was performed on an Illumina HiSeq 4000 (LC Sciences, USA). The average insert size for the paired-end library was 300 ± 50 bp. After quality control, a total of 47.5 million clean reads were obtained and assembled using the Trinity software (version 2.8.5). The assembled contigs were searched against NCBI virus RefSeqs (ftp://ftp.ncbi.nlm.nih.gov/refseq/release/viral) by the BLASTx algorithm with a cutoff E value of ≤10-5. 12 contigs sized from 3,421 to 4,093 bp were found to share a sequence identity of 77.5%-94.1% with SYCMV isolates from Japan (LC332541) and South Korea (JF495127.1). No other virus matches were identified. The largest contig (4,093 bp, MT816507) covers 99% of the expected complete genome of SYCMV (4,121 bp, KX096577). To verify the accuracy of the sequence assembled, RT-PCR-Sanger sequencing was performed on a single field plant sample using primers designed for SYCMV (Forward, 5'-GAACAAAGAGTCTGGATCTT-3'; Reverse, 5'-TCCTTCCAAAACCTCGCGGG-3'). The sequence of the amplicon (3854 bp, MT997092) exhibited an identity of 99.9% to the HTS-derived SYCMV contig sequence. Phylogenetic analysis of the amplicon sequence revealed that the SYCMV isolate from China formed a distinct branch in the tree (Fig. S1). Sap from symptomatic field plants was used to mechanically inoculate two soybean cultivars (Jiunong 9 and Kefeng 1, 10 plants per cultivar), and leaves inoculated with phosphate buffer saline (PBS, 0.01 M, pH 7.5) served as a control (3 plants per cultivar). All but the control plants developed systemic bright yellow mosaic symptoms 10 days after inoculation (Fig. S2A). The infection of the soybean plants with SYCMV was confirmed by RT-PCR with the newly designed primers for SYCMV (Forward, 5'- CCTACAGGCATTGGTTTCGT-3'; Reverse, 5'-CGTGAGGTTCTTGCTTCACA-3', anticipated amplicon size: 2,210 bp) (Fig. S2B) and by amplicon sequencing (100% sequence identity with MT9979092). In addition, the infection was further confirmed by immuno-blotting using an antibody against SYCMV coat protein (synthesized by GenScript, USA) (Fig. S2C). Together, the results demonstrate that SYCMV is the causal agent of the bright yellow mosaic symptoms in soybean observed in the field. To the best of our knowledge, this is the first report of SYCMV on soybean in China. These findings shall not only alert local growers to a potential new threat to soybean production in their region, but also provide new insights on the transmission, epidemiology and pathological properties of SYCMV in China.

First Report of Cowpea Mild Mottle Virus Infecting Soybean in China

Cowpea mild mottle virus (CPMMV) is a positive-strand RNA virus of the genus Carlavirus in the family Betaflexiviridae that is transmitted by the whitefly Bemisia tabaci in a nonpersistent manner. It was first reported to infect cowpea in Ghana (Brunt and Kenten 1973) and subsequently found in Fabaceae and other crops across the world (Laguna et al. 2006; Zanardo et al. 2014). CPMMV causes mosaic, dwarfism, and stem necrosis in soybean (Glycine max L. Merr.), one of the most important oil crops in the world. There are no confirmed reports or information about CPMMV on soybean in China. In September 2019, leaf mosaic and crinkling symptoms were observed on soybean (cv. Zhonghuang 33) during field surveys at three locations in Anhui Province, China. The disease incidence was estimated as 20 to 40% across the field. Leaves with typical virus-like symptoms were collected from five symptomatic plants and analyzed by high-throughput sequencing (HTS) to investigate the etiology. Total RNA was extracted by TRIzol Reagent (Invitrogen, U.S.A.), and approximately 10 μg of total RNA was used for transcriptome sequencing. The final cDNA library was constructed using the TruSeq Stranded mRNA Library Prep Kit (Illumina, U.S.A.) and sequenced on an Illumina HiSeq 4000 (LC Sciences, U.S.A.). The average insert size for the paired-end libraries was 300 bp (±50 bp). A total of 30,098,814 raw reads were obtained, and the reads were assembled using Trinity software (version 2.8.5). The assembled contigs were searched against NCBI virus RefSeqs (ftp://ftp.ncbi.nlm.nih.gov/refseq/release/viral) using a BlastX algorithm with a cutoff E value of ≤10⁻⁵. Thirty-two contigs were similar to CPMMV, which was the only plant virus (E value ≤10⁻⁵; contigs ≥200 nt) found in our HTS data. The largest contig of 8,256 bp shared 95.7% nucleotide identity to a CPMMV isolate (accession no. KY420906) sequenced from cowpea in Hainan, China. Phylogenetic analysis showed the isolate from China clustered separately from isolates from other countries. The sequence of the CPMMV isolate from Anhui was submitted to NCBI under the accession number MN908944. Mechanical transmission assays were performed using sap from symptomatic field plants and two soybean varieties (Williams 82 and Kefeng 1, 10 plants per variety). Eight of the Williams 82 plants and all Kefeng 1 plants showed systemic mosaic 14 days postinoculation, whereas control plants inoculated with phosphate-buffered saline (three per cultivar) were all symptomless. To confirm the transmission, an RT-PCR assay was performed on mechanically inoculated plants using primers designed to be specific for the CPMMV-coat protein (5′-CTGGCCAAGTGGTTTGTTTT-3′; 5′-AACCGGGTTTACAATCCACA-3′). The expected 1,865-bp PCR products were obtained from each of the five symptomatic plants tested but not from symptomless control plants. The PCR products were directly sequenced and were almost identical (99.8% nt) to the corresponding HTS-derived sequences of the isolate from Anhui province (MT232837). To the best of our knowledge, this is the first report of soybean infected by CPMMV in China. These findings show that CPMMV is a potentially important soybean disease in China that has implications for disease control and resistance breeding. This study is therefore an alert for local growers and those involved in disease management and will provide impetus and information for breeders in soybean disease resistance.

Construction and characterization of a BAC library for flue-cured tobacco line with high resistance to blank shank

<p indent="0mm">Tobacco (<italic>N. tabacum</italic>) is an important model crop in molecular biology research. In this study, a bacterial artificial chromosome (BAC) library of a flue-cured tobacco line 14–60 with high blank shank resistance and good quality was constructed. High molecular weight DNA was isolated using intact nuclei from tobacco, partially cleaved with <italic>Hin</italic>d III and cloned into the pIndigoBAC536-S vector. The BAC library consisted of 414,720 clones arrayed in one thousand and eighty 384-microtite plates, with an average insert size of 123 kb ranging from 97.0–145.5 kb. No empty insert clone was found. Based on an estimated genome size of 4500 Mb for common tobacco, the BAC library was estimated to cover 11 times of genome equivalents. The utility of the library was further confirmed by screening the library with the primers of tobacco <italic>hem</italic>A, <italic>NtFT</italic>, and<italic> eIF4E-</italic>1 genes. The high capacity library will serve as a giant genomic resource for map-based cloning of quantitative trait loci or genes associated with important agronomic and smoking quality traits or resistance to blank shank, physical mapping and comparative genome analysis.

BAC selection of the large white butterfly, Pieris brassicae (Lepidoptera: Pieridae) containing orthologs of the silkworm, Bombyx mori (Lepidoptera: Bombycidae)

We constructed a bacterial artificial chromosome (BAC) library from the large white butterfly, Pieris brassicae (L.) (Lepidoptera: Pieridae), which composed of 15,360 clones with an average insert size of 103 kb. We designed 39 degenerate oligonucleotide primer pairs and cloned the PCR products for sequencing. From these plus pierisin data, we designed single tagged sequence primers and selected 40 BAC clones using a 3-step PCR protocol. Additional 40 BACs were sequenced to improve identification of P. brassicae BACs carrying orthologs of the silkworm, Bombyx mori (L.) (Lepidoptera: Bombycidae). Among 34 BAC clones containing multiple B. mori orthologs, sequences in 27 BACs correspond to a block (less than 1 Mb) of every B. mori chromosome. While orthologs in 5 BACs showed collinearity to each B. mori chromosome with a distance (1.4–6.8 Mb), orthologs in two BACs corresponded to genes in more than two different B. mori chromosomes.

Abstract 745: Development of a comprehensive gene fusion NGS panel using an integrated microfluidic circuit enabling highly efficient, multiplex PCR enrichment

Abstract Introduction: RNA fusion transcripts result from genomic rearrangements where two distinct genes become juxtaposed and fused. Fusion transcripts are an important class of somatic alterations because they have the potential to create chimeric proteins with altered function, contributing to oncogene activation. These aberrant proteins are expressed in a tumor-specific manner and are thus excellent targets for therapeutic intervention. We have designed an amplicon-based library preparation (LP) kit that targets over 350 fusion gene pairs, representing over 1,000 unique fusion breakpoint events from both solid tumor and hematologic cancers. This panel is specifically designed to run on a newly developed microfluidic integrated fluidic circuit (IFC) that supports flexible amplicon library preparation of 1-6 unique LP panels on a single IFC. Samples can be run against the LP panels in groups of 8, allowing for up to 48 samples per IFC. In this poster we present the results of our analytical validation tests, demonstrating the performance of our comprehensive gene fusion panel on synthetic targets, reference FFPE samples, and subject FFPE samples for use in research studies. Methods: Primers for amplicon library generation targeting known fusion breakpoints were designed in collaboration with Q2 Solutions®. The panel is comprised of over 1,000 amplicons with an average insert size of 170 bp. Primers were divided into 8 assay pools using informatic parameters that minimize generation of off-target products. Assay pools and cDNA from an off-IFC reverse transcription reaction, along with sample barcodes, were dispensed into designated inlets on an IFC and placed in a Juno™ targeted DNA sequencing library preparation system for mixing the nanoscale reactions, thermal cycling, and amplicon harvesting. Harvested amplicons were collected from the IFC, pooled, and prepared for sequencing on an Illumina® NextSeq™. Results: The panel was tested against synthetic targets representing all targeted amplicons to demonstrate that each assay reliably detects the intended target of interest. Reference standards and subject FFPE samples were used to assess performance, including positive predictive agreement (PPA), positive predictive value (PPV), and limit of detection. Using these samples at inputs as low as 10 ng of total RNA, PPA and PPV were greater than 99% for the fusion events tested. In addition, fusion events could be reliably detected with as little as 250 copies of target fusion material loaded onto the IFC. Conclusions: A comprehensive fusion gene panel for targeted next-generation sequencing has been developed using nanoliter-scale PCR based enrichment on a newly developed IFC. Generation of high-quality libraries from a minimum of 10 ng total RNA from FFPE samples for use in research studies has been demonstrated. Citation Format: Michael L. Gonzales, Jian Qin, Xiaohui Wang, Sangpen Chamnongpol, Jeff Jasper, Thomas Goralski, Christopher Kubu. Development of a comprehensive gene fusion NGS panel using an integrated microfluidic circuit enabling highly efficient, multiplex PCR enrichment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 745.

Construction of a Genomic Bacterial Artificial Chromosome (BAC) Library for the Prawn Macrobrachium rosenbergii and Initial Analysis of ZW Chromosome-Derived BAC Inserts.

Knowledge on sex determination has proven valuable for commercial production of the prawn Macrobrachium rosenbergii due to sex dimorphism of the male and female individuals. Previous studies indicated that prawn sex is determined by a ZW-ZZ chromosomal system, but no genomic information is available for the sex chromosome. Herein, we constructed a genomic bacterial artificial chromosome (BAC) library and identified the ZW-derived BAC clones for initial analysis of the sex chromosomal DNA sequence. The arrayed BAC library contains 200,448 clones with average insert size of 115.4kb, corresponding to ∼ 4× coverage of the estimated 5.38Gb genome. Based on a short female-specific marker, a Z- and a W-fragment were retrieved with the genomic walking method. Screening the BAC library using a ZW-specific marker as probe resulted in 12 positive clones. From these, a Z-derived (P331M17) and a W-derived (P122G2) BAC clones were randomly selected and sequenced by PacBio method. We report the construction of a large insert, deep-coverage, and high-quality BAC library for M. rosenbergii that provides a useful resource for positional cloning of target genes, genomic organization, and comparative genomics analysis. Our study not only confirmed the ZW/ZZ system but also discovered sex-linked genes on ZW chromosomes for the first time, contributing to a comprehensive understanding of the genomic structure of sex chromosomes in M. rosenbergii.

Molecular marker development and primary physical map construction for the tuber shape Ro gene locus in diploid potato (Solanum tuberosum L.)

Potato (Solanum tuberosum L.) is an important crop worldwide that is consumed fresh and processed in many different ways. The tuber shape is a critical trait in potato breeding, because it directly affects processing quality. Previous studies have demonstrated that the Ro locus on chromosome X is the major locus controlling tuber shape, and the round shape allele, Ro, is dominant over the long shape allele, ro. However, no reports on the cloning or physical mapping of this locus have been published. In this study, whole genome sequencing and bacterial artificial chromosome (BAC) library construction were applied to develop molecular markers and construct a physical map of Ro. Eight round-tuber genotypes were used to construct the BAC library, which contains about 40,000 clones with an average insert size of 85 kb and represents approximately four haploid potato genomes. A total of 66 individuals with extreme round or long tubers were selected to construct a round-tuber shape DNA bulk and a long-tuber shape DNA bulk for WGS. Single-nucleotide polymorphisms (SNPs) identified from WGS data were used to develop nine polymorphic markers linked to Ro. These markers were used to screen the BAC library, and eight positive BAC clones were ultimately identified and then analyzed by restriction enzyme digestion and BAC end sequencing and used to develop markers and identify new positive BAC clones. Finally, a primary physical map of Ro was constructed using a chromosome walking strategy. This map provides an important reference for cloning the potato tuber shape gene and studying tuber shape variation.

Generation and characterization of expressed sequence tags (ESTs) from coralloid root cDNA library of Cycas debaoensis

A normalized full-length cDNA library was constructed from the coralloid roots of Cycas debaoensis by the DSN (duplex-specific nuclease) normalization method combined with the SMART (Switching Mechanism At 5′ end of the RNA Transcript) technique. The titer of the original cDNA library was about 1.5 × 106 cfu·mL−1 and the average insertion size was about 1 kb with a high recombination rate (97%). The 5011 high-quality expressed sequence tags (ESTs) were obtained from 5393 randomly picked cDNA clones. Clustering and assembly of ESTs resulted in 2984 unique sequences, consisting of 618 contigs and 2366 singlets. EST sequence annotation revealed that 2333 and 1901 unigenes were functionally annotated in the NCBI non-redundant database and Swiss-Prot protein database, respectively. Functional analysis demonstrated that 1495 (50.1%) unigenes were associated with 4082 Gene Ontology (GO) terms. A total of 847 unigenes were grouped into 22 Cluster of Orthologous Groups (COG) functional categories. Based on the EST dataset, 22 ESTs that encoded putative receptor-like protein kinase (RLK) genes were screened. Furthermore, a total of 94 simple sequence repeats (SSRs) were discovered, of which 20 loci were successfully amplified in C. debaoensis. This study is the first EST analysis for the coralloid roots of C. debaoensis and provides a valuable genomic resource for novel gene discovery, gene expression and comparative genomics, conservation and management studies as well as applications in C. debaoensis and related cycad species.

An assessment of transgenomics as a tool for gene discovery in Populus euphratica Oliv.

Transgenomics for gene discovery in Populus euphratica. Transgenomics, a member of the omics family of methodologies, is characterized as the introduction of DNA from one organism into another on a genome-wide scale followed by the identification of recipients with altered phenotypes. This strategy allows investigators to identify the gene(s) involved in these phenotypic changes. It is particularly promising for woody plants that have a long life cycle and for which molecular tools are limited. In this study, we constructed a large-insert binary bacterial artificial chromosome library of Populus euphratica, a stress-tolerant poplar species, which included 55,296 clones with average insert sizes of about 127kb. To date, 1077 of the clones have been transformed into Arabidopsis thaliana via Agrobacterium by the floral dip method. Of these, 69 transgenic lines showed phenotypic changes represented by diverse aspects of plant form and development, 22 of which were reproducibly associated with the same phenotypic change. One of the clones conferring transgenic plants with increased salt tolerance, 002A1F06, was further analyzed and the 127,284bp insert in this clone harbored eight genes that have been previously reported to be involved in stress resistance. This study demonstrates that transgenomics is useful in the study of functional genomics of woody plants and in the identification of novel gene(s) responsible for economically important traits. Thus, transgenomics can also be used for validation of quantitative trait loci mapped by molecular markers.

Abstract 4651: Development of a targeted NGS panel for solid tumor actionable gene targets using multiplex PCR-based enrichment in an integrated fluidic circuit

Abstract Introduction: Next-generation sequencing (NGS) has been being rapidly adopted in clinical research to align actionable variants in tumors to targeted therapies or clinical trials. Comprehensive NGS-based tumor profiling assays are an efficient and effective method to characterize a variety of clinically relevant somatic mutations. We have developed a targeted NGS cancer gene panel that focuses on actionable gene mutations and employs a NGS library preparation method based on multiplex PCR enrichment in an integrated fluidic circuit (IFC) and the automated JunoTM System. Methods: 53 actionable cancer genes were selected based on both clinical and research knowledge that covers 15 solid tumor types. Assays for the selected regions of the 53 genes were designed by an internal assay design pipeline. Multiplex PCR using target-specific primers is conducted in an LP—48.48 IFC on the Fluidigm Juno™ system, where up to 48 DNA samples can be processed simultaneously. A unique barcode is incorporated in one of the PCR primers to distinguish individual samples. To evaluate the assay performance, three sets of cell line gDNA samples were identified from commercial sources: set 1: 22 commercial reference samples, set 2: 12 samples for single nucleotide variants (SNVs) and indels, and set 3: 12 samples for copy number variations (CNVs). Each sample was tested on the IFC in 4 replicates. PCR products harvested from the IFC were pooled and purified with Agencourt® AMPure® XP magnetic beads. A second PCR step using a universal primer pair was performed to add sequencing adapters. After a second purification, the DNA library was sequenced on a NextSeq™ 500 system. The data analysis was performed by a service provider partner (GenomOncology). Results: A total of 1,508 primer pairs with an average insert size of 155 bp were selected to cover SNVs, indels, and CNVs. The assays were separated into 44 pools to minimize the interaction between assay primers and improve performance. The percentage of reads mapped to the genome was 98.9%, and the percentage of reads that mapped to amplicons was 96.8%. Mutation detection sensitivity was 4% variant allele frequency (VAF.) In 47 selected samples, the total SNVs, CNVs and indels represented are 182, 154 and 28, respectively. For SNVs, the positive percent agreement (PPA) is 1.0 and positive predictive value (PPV) is 0.974. For CNVs, the PPA is 1.0 and PPV is 0.969 and for indels, the PPA and PPV are 1.0 and 0.966, respectively. Overall concordance is 0.99. Conclusions: A targeted NGS cancer panel employing PCR-based enrichment on an IFC has been developed and yielded high quality of libraries generated on the JunoTM system for detecting SNVs, indels and CNV in solid tumor samples. This panel covers actionable targets in 53 cancer genes and utilizes a microfluidic device to provide a simple streamlined workflow for library preparation of up to 48 DNA samples per IFC. Citation Format: Peilin Chen, Jaibiao Gong, David Wang, Devin Do, Lianne McLean, Tom Goralski. Development of a targeted NGS panel for solid tumor actionable gene targets using multiplex PCR-based enrichment in an integrated fluidic circuit [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4651.

Chloramphenicol Derivatives with Antibacterial Activity Identified by Functional Metagenomics

A functional metagenomic approach identified novel and diverse soil-derived DNAs encoding inhibitors to methicillin-resistant Staphylococcus aureus (MRSA). A metagenomic DNA soil library containing 19 200 recombinant Escherichia coli BAC clones with 100 Kb average insert size was screened for antibiotic activity. Twenty-seven clones inhibited MRSA, seven of which were found by LC-MS to possess modified chloramphenicol ( Cm) derivatives, including three new compounds whose structures were established as 1-acetyl-3-propanoylchloramphenicol, 1-acetyl-3-butanoylchloramphenicol, and 3-butanoyl-1-propanoylchloramphenicol. Cm was used as the selectable antibiotic for cloning, suggesting that heterologously expressed enzymes resulted in derivatization of Cm into new chemical entities with biological activity. An esterase was found to be responsible for the enzymatic regeneration of Cm, and the gene trfA responsible for plasmid copy induction was found to be responsible for inducing antibacterial activity in some clones. Six additional acylchloramphenicols were synthesized for structure and antibacterial activity relationship studies, with 1- p-nitrobenzoylchloramphenicol the most active against Mycobacterium intracellulare and Mycobacterium tuberculosis, with MICs of 12.5 and 50.0 μg/mL, respectively.

Construction and characterization of a bacterial artificial chromosome library for Gossypium mustelinum.

A bacterial artificial chromosome (BAC) library for G. mustelinum Miers ex G. Watt (AD4) was constructed. Intact nuclei from G. mustelinum (AD4) were used to isolate high molecular weight DNA, which was partially cleaved with Hind III and cloned into pSMART BAC (Hind III) vectors. The BAC library consisted of 208,182 clones arrayed in 542 384-microtiter plates, with an average insert size of 121.72 kb ranging from 100 to 150 kb. About 2% of the clones did not contain inserts. Based on an estimated genome size of 2372 Mb for G. mustelinum, the BAC library was estimated to have a total coverage of 10.50 × genome equivalents. The high capacity library of G. mustelinum will serve as a giant gene resource for map-based cloning of quantitative trait loci or genes associated with important agronomic traits or resistance to Verticillium wilt, physical mapping and comparative genome analysis.

Reconstruction of the repetitive antifreeze glycoprotein genomic loci in the cold-water gadids Boreogadus saida and Microgadus tomcod

Antifreeze glycoproteins (AFGPs) are a novel evolutionary innovation in members of the northern cod fish family (Gadidae), crucial in preventing death from inoculative freezing by environmental ice in their frigid Arctic and sub-Arctic habitats. However, the genomic origin and molecular mechanism of evolution of this novel life-saving adaptive genetic trait remained to be definitively determined. To this end, we constructed large insert genomic DNA BAC (bacterial artificial chromosome) libraries for two AFGP-bearing gadids, the high-Arctic polar cod Boreogadus saida and the cold-temperate Atlantic tomcod Microgadus tomcod, to isolate and sequence their AFGP genomic regions for fine resolution evolutionary analyses. The BAC library construction encountered poor cloning efficiency initially, which we resolved by pretreating the agarose-embedded erythrocyte DNA with a cationic detergent, a method that may be of general use to BAC cloning for teleost species and/or where erythrocytes are the source of input DNA. The polar cod BAC library encompassed 92,160 clones with an average insert size of 94.7 kbp, and the Atlantic tomcod library contained 73,728 clones with an average insert size of 89.6 kbp. The genome sizes of B. saida and M. tomcod were estimated by cell flow cytometry to be 836 Mbp and 645 Mbp respectively, thus their BAC libraries have approximately 10- and 9.7-fold genome coverage respectively. The inclusiveness and depth of coverage were empirically confirmed by screening the libraries with three housekeeping genes. The BAC clones that mapped to the AFGP genomic loci of the two gadids were then isolated by screening the BAC libraries with gadid AFGP gene probes. Eight minimal tiling path (MTP) clones were identified for B. saida, sequenced, and assembled. The B. saida AFGP locus reconstruction produced both haplotypes, and the locus comprises three distinct AFGP gene clusters, containing a total of 16 AFGP genes and spanning a combined distance of 512 kbp. The M. tomcod AFGP locus is much smaller at approximately 80 kbp, and contains only three AFGP genes. Fluorescent in situ hybridization with an AFGP gene probe showed the AFGP locus in both species occupies a single chromosomal location. The large AFGP locus with its high gene dosage in B. saida is consistent with its chronically freezing high Arctic habitats, while the small gene family in M. tomcod correlates with its milder habitats in lower latitudes. The results from this study provided the data for fine resolution sequence analyses that would yield insight into the molecular mechanisms and history of gadid AFGP gene evolution driven by northern hemisphere glaciation.

Construction and characterization of a bacterial artificial chromosome library for Camellia sinensis

Tea is a popular and natural non-alcoholic beverage, and is produced from fresh leaves of Camellia sinensis. Tea leaves contain many bioactive compounds that have significant health benefits. We constructed a high quality bacterial artificial chromosome (BAC) library by using the fresh petals of C. sinensis “Shuchazao” for genome sequencing and improvement of genomic assembly. BAC library is still a significant tool for studies of functional genomes and preservation of precious genetic resources. The BAC library contains 161,280 clones with an average insert size of 113 kb, which represents approximately 6.2-fold coverage of haploid genome equivalents of C. sinensis. We characterized 20 complete BAC clones and 738 BAC end sequences (BESs) ranging from 105 to 917 bp. In addition, we predicted cis-regulatory elements of LAR (leucoanthocyanidin reductase), TCS (caffeine synthase), and TS (theanine synthetase) involved in tea characteristic metabolite synthesis and identified a larger number of light-responsive cis-acting elements in these three genes. Meanwhile, we analyzed alternative splicing of these three genes. Furthermore, 12 pairs of SSR primers were successfully amplified in tea plant DNA. The tea BAC library was a critical resource to accomplish de novo whole-genome sequencing, accelerate gene discovery and enhance molecular breeding of C. sinensis.

Abstract 738: Highly multiplexed and precisely calibrated reference materials for copy number variation detection

Abstract Introduction: Genomic instability is a hallmark of cancer, and copy number variation (CNV), including gene duplication and deletion, can be a key driver of oncogenesis. Detection of CNV is needed for personalized treatment, and may involve microarray analysis or Next Generation Sequencing. Often, DNA from patient-derived cell lines is used as positive control material in these assays. However, these materials are often poorly characterized, may change over time, and have additional confounding genetic alterations. Here we describe the development of new reference materials that are highly characterized by digital PCR and can aid in the validation and calibration of new diagnostic assays for CNV. Methods: The GM24385 reference cell line has been extensively characterized by the Genome in a Bottle project1 and originates from a participant in the Personal Genome Project. DNA from this cell line was used to make a library of full-length gene inserts. The library had &amp;gt;12X coverage of the GM24385 genome and an average insert size of ~135 Kb. PCR based screening was used to identify and isolate full length MYC, FGFR3 and ERBB2 genes. Next Generation Sequencing, using Illumina Nextera library preparation kits and a MiSeq instrument, was used to verify the gene of interest. The genomic DNA and gene inserts were then formulated into reference materials, in purified DNA format to amplify the copy numbers of these key genes. Digital PCR was used to control the mixing and verify the number of copies of genes and/or mutations within the reference material. Results: The sequence confirmed genes were formulated into GM24385 genomic DNA at levels consistent with 5X and 10X amplification. Testing on the ArcherDx VariantPlex™ Solid Tumor Kit indicated that each amplification could be detected at the expected level. Conclusions: Highly characterized and consistently manufactured reference materials for copy number detection are currently lacking. The described Seraseq CNV Reference Materials provide proof of concept for a biosynthetic, engineered material that uses digital PCR as an orthologous reference method and provides a “ground truth” copy number level for key oncogenes. Citation Format: Catherine Huang, Yves Konigshofer, Jessica Dickens, Bharathi Anekella. Highly multiplexed and precisely calibrated reference materials for copy number variation detection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 738. doi:10.1158/1538-7445.AM2017-738

Bacterial artificial chromosome library construction of root-knot nematode resistant pepper genotype HDA149 and identification of clones linked to Me3 resistant locus

Pepper (Capsicum annuum. L.) is a widely cultivated vegetable crop worldwide and has the second largest planting area and the first largest vegetable output and value in China. Pepper root-knot nematode (Meloidogyne spp.) is one of the most serious pests of pepper, which caused huge losses every year. Previous studies showed that the Me3 gene is resistant to a wide range of Meloidogyne species, including M. arenaria, M. javanica, and M. incognita. HDA149, a double haploid pepper genotype, harboring the root-knot nematode resistance gene Me3, was used to construct bacterial artificial chromosome library (BAC) via the vector of CopyControl™ pCC1 in this study. The library consists of 210 200 BAC clones and is equivalent to 5.3 pepper genomes. The average insert size is 95 kb, and most of them are 90–120 kb; but the empty clones are less than 3%. In order to screen the BAC library easily, 550 super pools with 384 BAC clones of each pool were further developed in this study. Specific primers from Me3 gene locus were used for BAC library screening, and more than 20 positive BAC clones were obtained. Then the selected positive BAC clones were analyzed by restriction enzyme digestion, BAC-end sequencing, marker development, and new positive BAC clones exploration, respectively. Finally, the contig with total length of about 300 kb linked to the Me3 locus was constructed based on chromosome walking strategy, which made a solid foundation for the cloning of the important root-knot nematode resistance gene Me3.

A genome-wide BAC-end sequence survey provides first insights into sweetpotato (Ipomoea batatas (L.) Lam.) genome composition.

BackgroundSweetpotato, Ipomoea batatas (L.) Lam., is an important food crop widely grown in the world. However, little is known about the genome of this species because it is a highly heterozygous hexaploid. Gaining a more in-depth knowledge of sweetpotato genome is therefore necessary and imperative. In this study, the first bacterial artificial chromosome (BAC) library of sweetpotato was constructed. Clones from the BAC library were end-sequenced and analyzed to provide genome-wide information about this species.ResultsThe BAC library contained 240,384 clones with an average insert size of 101 kb and had a 7.93–10.82 × coverage of the genome, and the probability of isolating any single-copy DNA sequence from the library was more than 99%. Both ends of 8310 BAC clones randomly selected from the library were sequenced to generate 11,542 high-quality BAC-end sequences (BESs), with an accumulative length of 7,595,261 bp and an average length of 658 bp. Analysis of the BESs revealed that 12.17% of the sweetpotato genome were known repetitive DNA, including 7.37% long terminal repeat (LTR) retrotransposons, 1.15% Non-LTR retrotransposons and 1.42% Class II DNA transposons etc., 18.31% of the genome were identified as sweetpotato-unique repetitive DNA and 10.00% of the genome were predicted to be coding regions. In total, 3,846 simple sequences repeats (SSRs) were identified, with a density of one SSR per 1.93 kb, from which 288 SSRs primers were designed and tested for length polymorphism using 20 sweetpotato accessions, 173 (60.07%) of them produced polymorphic bands. Sweetpotato BESs had significant hits to the genome sequences of I. trifida and more matches to the whole-genome sequences of Solanum lycopersicum than those of Vitis vinifera, Theobroma cacao and Arabidopsis thaliana.ConclusionsThe first BAC library for sweetpotato has been successfully constructed. The high quality BESs provide first insights into sweetpotato genome composition, and have significant hits to the genome sequences of I. trifida and more matches to the whole-genome sequences of Solanum lycopersicum. These resources as a robust platform will be used in high-resolution mapping, gene cloning, assembly of genome sequences, comparative genomics and evolution for sweetpotato.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-3302-1) contains supplementary material, which is available to authorized users.