title: Invertebrate sample from Tenuignathia rikerae
organism: Tenuignathia rikerae
description: For the gnathostomulid Tenuignathia rikerae, we attempted a genome assembly from DNA amplified from a single individual by multiple displacement amplification. In brief, a single RNAlater preserved specimen was rinsed in distilled water, and kept in a lens of ca. 2 uL, to which alkaline lysis buffer from the Qiagen REPLI-g Single-Cell Kit (Qiagen, Inc., cat. # 150343) was directly added, and DNA amplified as per manufacturer's recommendations with the single-cell protocol, incubating at 30 C for 8 hours. MDA product was QC'd and confirmed to be HMW (70 kbp+) on an Agilent Genomic DNA TapeStation 2200 kit (Agilent, Inc. cat. # 5067-5365). One aliquot was taken and fragmented to ~350 bp using a Covaris S220 focused ultrasonicator and prepared as an Illumina short-insert genomic DNA library using a previously described protocol for end-repair/A-tailing and ligation of Y-shaped adapters (Neiman et al., 2012), followed by limited library amplification with indexing primers and KAPA HiFi polymerase (KK2103). 1 ug of MDA product was processed into a mate-pair library using the Illumina Nextera Mate-Pair Library Preparation Kit (Illumina, Inc. cat. # FC-132-1001), following the manufacturer's gel-free protocol, and amplifying the final Illumina library with indexing oligos and the KAPA HiFi polymerase. Adapter sequences were trimmed from the mate-pair library using NextClip (Leggett et al., 2014) using default parameters, and from the short-insert library using Trimmomatic as described above. An initial assembly using SPAdes v3.2.0 was created running in single-cell mode; however, the assembly failed to finish at the k-mer size step of 55. We therefore took the error-corrected reads output by SPAdes, and used these as inputs for assembly with IDBA_UD v1.1.1 (Peng et al., 2012). The resulting assembly remained highly fragmented (1,691,903 scaffolds of which 1,140,751 were <200 bp, with a 496.07 Mbp span, a scaffold N50 of 360 bp, and GC% of 32.25). Nonetheless, we conducted an ab initio gene prediction effort on the resultant assembly, using Augustus v3.2.2, and though still highly incomplete (e.g. with only 37.29% and 61.39% of complete and complete+partial BUSCO v3 eukaryotic core genes, respectively), these peptides were still judged to be a sufficiently large sample to be useful in the context of phylogenetic inference.
attributes.tag: isolate
attributes.value: Wild-caught isolate
attributes.tag: isolation_source
attributes.value: Bermuda, collected Wolfgang Sterrer
attributes.tag: collection_date
attributes.value: Unknown
attributes.tag: geo_loc_name
attributes.value: Bermuda
attributes.tag: tissue
attributes.value: Whole-organism
attributes.tag: BioSampleModel
attributes.value: Invertebrate