Low-coverage whole genome raw sequence reads from two ostariophysan lineages.

We purchased specimens of Apteronotus albifrons and Corydoras paleatus from commercial wholesalers in the Los Angeles, CA area, collected tissues following protocols approved by the University of California Los Angeles Institutional Animal Care and Use Committee (Approval 2008-176-21), and extracted DNA from each tissue using a commercial kit following the manufacturer's instructions (DNeasy, Qiagen N.V.). After extraction, we quantified 2 µL of DNA using a Qubit fluorometer (Invitrogen, Inc.) following the manufacturer''s protocol, and we visualized 50-100 ng of each extract by electrophoresis through 1.5% (w/v) agarose gel in TBE or TAE. Following this quality check, we prepared 100 µL (~10 ng/µL) aliquots of extracted DNA, and we sheared each sample to 300-600 bp in length using 5-10 cycles of sonication (High; 30 s on; 90 s off) on a BioRuptor (Diagenode, Inc.). We prepared single-indexed sequencing libraries from 0.5-1.0 µg sheared DNA extracts using a commercial library preparation kit (Kapa Biosystems, Inc.) and a set of custom-indexed sequencing adapters (Faircloth and Glenn, 2012). Following library preparation, we size-selected the Corydoras library to span a range of 200-300 bp using agarose-gel-based size selection. We did not size-select the Apteronotus library. We amplified both libraries using 6-10 cycles of PCR, and we purified library amplifications using SPRI beads (Rohland and Reich, 2012). Following purification, we checked the insert size distribution of each library using a BioAnalyzer (Agilent, Inc.), and we quantified libraries using a commercial qPCR quantification kit (Kapa Biosystems, Inc.). We ran each library on a separate lane of Illumina, paired-end, 100 bp sequencing (PE100) by combining each library into a pool of unrelated (and differently indexed) samples, and we sequenced each library pool using an Illumina HiSeq 2500 at the UCLA Neuroscience Genomics Core (UNGC). We demultiplexed the sequencing data using bcl2fastq 1.8.4 and allowing one base pair mismatches between the expected and observed indexes (the index sequences we used were robust to = 3 insertion, deletion, or substitution errors).We validated the species identification of each sample by aligning FASTQ reads to a related mtDNA genome using bwa mem v0.7.17 (Li, 2013), reducing the resulting BAM file to aligning reads using samtools v0.1.18 (Li et al., 2009), and converting the BAM file of aligned reads back to paired FASTQ reads using bedtools v2.17.0 (Quinlan and Hall, 2010). We assembled the resulting FASTQ data using spades v3.10.1 (Nurk et al., 2013) with read correction, a kmer length of 55, and the `--careful` assembly option. From the contig that resulted (which was either equal to or slightly shorter than the general mtDNA sequence length for vertebrates), we used a program within the phyluce package (Faircloth, 2015) to extract the portions of each contig that were similar to COI sequences from Apteronotus (NCBI GenBank AB054132.1:5453-7012) and Corydoras (NCBI GenBank JN988809.1). We then matched these extracted COI sequences against the Species Level Barcode Records in the BOLD Systems Database (http://www.boldsystems.org; search performed August 2018). For Apteronotus albifrons, the top hit (100% sequence identity) validated the species identification, and for Corydoras sp., the top publicly available hit (99.85% sequence identity) was Corydoras paleatus (NCBI GenBank JX111734.1; (Rosso et al., 2012)).我们从加利福尼亚州洛杉矶地区的商业批发商处购得白吻长颌电鳗（Apteronotus albifrons）和淡色兵鲶（Corydoras paleatus）的标本，按照加州大学洛杉矶分校动物护理与使用委员会（University of California Los Angeles Institutional Animal Care and Use Committee，审批编号2008-176-21）批准的实验方案采集组织样本，并使用商业试剂盒（DNeasy，凯杰公司（Qiagen N.V.））按照制造商说明书从每份组织中提取脱氧核糖核酸（DNA）。提取完成后，我们取2微升DNA样本，使用Qubit荧光计（Invitrogen公司）按照制造商提供的实验方案进行定量检测；同时取每份提取物50-100纳克，在含1.5%（质量体积比）琼脂糖的TBE或TAE缓冲液中进行琼脂糖凝胶电泳，以可视化检测DNA完整性。完成质量质控后，我们将提取的DNA制备为100微升（约10纳克/微升）的等分试样，使用Diagenode公司的BioRuptor超声破碎仪，以5-10次循环（高强度模式；超声30秒，间歇90秒）将每份样本的DNA片段化至300-600碱基对。我们使用商业文库制备试剂盒（Kapa Biosystems公司）和定制索引测序接头（Faircloth与Glenn，2012），从0.5-1.0微克的片段化DNA提取物制备单索引测序文库。文库构建完成后，我们采用琼脂糖凝胶筛选法将淡色兵鲶文库的插入片段大小富集至200-300碱基对，而未对白吻长颌电鳗文库进行大小筛选。我们通过6-10轮聚合酶链式反应（PCR）扩增两份文库，并使用SPRI磁珠（Rohland与Reich，2012）纯化文库扩增产物。纯化完成后，我们使用安捷伦（Agilent）公司的BioAnalyzer检测每份文库的插入片段长度分布，并使用商业定量聚合酶链式反应（qPCR）试剂盒（Kapa Biosystems公司）对文库进行定量。我们将每份文库与其他非相关（且索引序列不同）的样本混合成文库池，随后在Illumina HiSeq 2500平台上，于加州大学洛杉矶分校神经基因组学核心实验室（UNGC）进行Illumina双端100碱基对测序（PE100），每份文库单独占用一个测序泳道。我们使用bcl2fastq 1.8.4对测序数据进行样本拆分，允许预期与观测到的索引序列之间存在1个碱基错配（本次使用的索引序列可耐受至多3个插入、缺失或替换错误）。我们通过以下步骤验证每份样本的物种身份：将FASTQ格式的测序reads比对至相关线粒体DNA（mtDNA）基因组，使用bwa mem v0.7.17（Li，2013）；使用samtools v0.1.18（Li等人，2009）将比对生成的BAM文件过滤为仅保留成功比对的reads；使用bedtools v2.17.0（Quinlan与Hall，2010）将过滤后的BAM文件转换回双端FASTQ格式的reads。我们使用spades v3.10.1（Nurk等人，2013）对得到的FASTQ数据进行基因组组装，开启reads校正模式，设置kmer长度为55，并启用`--careful`组装参数。从得到的重叠群（contig，其长度与脊椎动物通用线粒体DNA序列长度相当或略短）中，我们使用phyluce软件包（Faircloth，2015）中的工具，提取每份重叠群中与白吻长颌电鳗（NCBI GenBank AB054132.1:5453-7012）和淡色兵鲶（NCBI GenBank JN988809.1）的细胞色素C氧化酶亚基I（COI）序列相似的区域。随后我们将这些提取得到的COI序列与BOLD系统数据库（http://www.boldsystems.org；检索时间为2018年8月）中的物种级条形码记录进行比对。对于白吻长颌电鳗，最佳比对结果（序列一致性100%）验证了其物种身份；对于兵鲶属（Corydoras sp.）样本，公开数据库中最佳比对结果（序列一致性99.85%）为淡色兵鲶（NCBI GenBank JX111734.1；Rosso等人，2012）。