A High-Quality Reference Genome for the Invasive Mosquitofish Gambusia affinis Using a Chicago Library

The western mosquitofish, Gambusia affinis, is a freshwater poecilid fish native to the southeastern United States but with a global distribution due to widespread human introduction. Gambusia affinis has been used as a model species for a broad range of evolutionary and ecological studies. We sequenced the genome of a male G. affinis to facilitate genetic studies in diverse fields including invasion biology and comparative genetics. We generated Illumina short read data from paired-end libraries and in vitro proximity-ligation libraries. We obtained 54.9× coverage, N50 contig length of 17.6 kb, and N50 scaffold length of 6.65 Mb. Compared to two other species in the Poeciliidae family, G. affinis has slightly fewer genes that have shorter total, exon, and intron length on average. Using a set of universal single-copy orthologs in fish genomes, we found 95.5% of these genes were complete in the G. affinis assembly. The number of transposable elements in the G. affinis assembly is similar to those of closely related species. The high-quality genome sequence and annotations we report will be valuable resources for scientists to map the genetic architecture of traits of interest in this species

Supplemental Material for Hoffberg et al., 2018

Figure S1: Comparison of the size distribution of library inserts in the Meraculous and HiRise assemblies.<div><br></div><div>Figure S2: The frequency of kmers at each kmer length. </div><div><br></div><div>Figure S3: The distribution of scaffold lengths in the HiRise assembly. </div><div><br></div><div>Figure S4: The cumulative percent of the assembly for a given scaffold size in the Meraculous and HiRise assemblies. </div><div><br></div><div>Table S1: A detailed list of the number of copies and percent of the assembly of transposons and repeatable elements. </div><div><br></div><p>File S1: Submission script for MAKER.</p><p><br></p> <p>File S2: MAKER executable file (maker_exe.ctl).</p><p><br></p> <p>File S3: Specifications for downstream filtering of BLAST and Exonerate alignments (maker_bopts.ctl).</p><p><br></p> <p>File S4: Primary configuration of MAKER specific options (maker_opts.ctl).</p><p><br></p> <p>File S5: Commands for training SNAP.</p> <p><br></p><p>File S6: Submission script for BLAST comparing <i>Gambusia affinis</i> with related fish.</p> <p><br></p><p>File S7: Submission script for BUSCO.</p> <p><br></p><p>File S8: Submission script for predicting ncRNAs.</p> <p><br></p><p>File S9: Illumina reads mapped to the reference in BAM format.</p><p><br></p><p>File S10: Sequence of tRNAs.</p> <p><br></p><p>File S11: Structure of tRNAs.</p> <div><br></div><div>File S12: rRNA, snRNA, snoRNA, and miRNA sequences.</div><div><br></div

Adapterama II: universal amplicon sequencing on Illumina platforms (TaggiMatrix)

Next-generation sequencing (NGS) of amplicons is used in a wide variety of contexts. In many cases, NGS amplicon sequencing remains overly expensive and inflexible, with library preparation strategies relying upon the fusion of locus-specific primers to full-length adapter sequences with a single identifying sequence or ligating adapters onto PCR products. In Adapterama I, we presented universal stubs and primers to produce thousands of unique index combinations and a modifiable system for incorporating them into Illumina libraries. Here, we describe multiple ways to use the Adapterama system and other approaches for amplicon sequencing on Illumina instruments. In the variant we use most frequently for large-scale projects, we fuse partial adapter sequences (TruSeq or Nextera) onto the 5′ end of locus-specific PCR primers with variable-length tag sequences between the adapter and locus-specific sequences. These fusion primers can be used combinatorially to amplify samples within a 96-well plate (8 forward primers + 12 reverse primers yield 8 × 12 = 96 combinations), and the resulting amplicons can be pooled. The initial PCR products then serve as template for a second round of PCR with dual-indexed iTru or iNext primers (also used combinatorially) to make full-length libraries. The resulting quadruple-indexed amplicons have diversity at most base positions and can be pooled with any standard Illumina library for sequencing. The number of sequencing reads from the amplicon pools can be adjusted, facilitating deep sequencing when required or reducing sequencing costs per sample to an economically trivial amount when deep coverage is not needed. We demonstrate the utility and versatility of our approaches with results from six projects using different implementations of our protocols. Thus, we show that these methods facilitate amplicon library construction for Illumina instruments at reduced cost with increased flexibility. A simple web page to design fusion primers compatible with iTru primers is available at: http://baddna.uga.edu/tools-taggi.html. A fast and easy to use program to demultiplex amplicon pools with internal indexes is available at: https://github.com/lefeverde/Mr_Demuxy