PacBio Isoform Sequencing

Total RNA was extracted from pitcher tissues using the modified cetyltrimethylammonium bromide (CTAB) protocol (Abdul-Rahman et al., 2017). The quality and integrity of extracted total RNA were determined using Nanodrop ND-1000 (Thermo Fisher Scientific Inc., United States) and Agilent 2,100 bioanalyzer (Agilent Technologies, United States), respectively. Total RNA with RNA integrity number (RIN) >8 was submitted for library preparation and sequencing at Icahn Medical Institute (Mount Sinai, New York City, United States). One replicate per species with the highest RIN was chosen for sequencing. Full-length cDNAs were prepared using SMARTer PCR cDNA synthesis kit (Clontech) according to the manufacturer’s protocols. Double-stranded cDNAs were subjected to size selection using BluePippin (Sage Science, MA, United States) at the MW range of 1–3 kb. The PCR amplification profile was 95°C for 2 min, 15 cycles × (98°C for 20 s, 65°C for 15 s, 72°C for 4 min), and a final extension at 72°C for 5 min. Due to low yield after selection, N. ampullaria sample was further amplified for nine cycles (98°C for 20 s, 65°C for 15 s, 72°C for 1 min 45 s) and was size-selected again before preparing the SMRTbell library with a minimum of 1 μg of dsDNA based on the manufacturer’s SMRTbell template protocol (SMRTbell Template Preparation Kit 1.0). The SMRTbell libraries were purified by two sequential 0.45 × AMPure PB purifications (Pacific Biosciences) after exonuclease digestion of incomplete SMRTbell templates. Libraries were quantified by fluorimetry and assayed for quantity and size distribution by Bioanalyzer. A single SMRTbell library for individual species was sequenced using SMRT Cell v3 with P6-C4 chemistry on the PacBio RS II platform (Pacific Biosciences), each at a concentration of 110 pM (Zulkapli et al., 2017).

Subreads <300-bp and reads with quality <0.75 (corresponding to a predicted error rate of >25%) were filtered out. Sub-reads were filtered and subjected to circular consensus sequence (CCS) read analysis using PacBio SMRT Analysis Server v2.3.0 following the RS_IsoSeq protocol. In brief, cDNA primer and poly-A tails were removed and the read of inserts (ROIs) were classified into full-length and non-full-length. Iterative clustering for error correction (ICE) algorithm was also used and quiver polishing was performed to generate consensus isoform sequences at a high QV value of 0.99 and expected size of 1–2 kb. For the reference transcriptome, raw reads from all three species were combined for the CCS read analysis.

Raw sequences were deposited into the Sequence Read Archive (SRA) under BioProject PRJNA299862 with the following identifiers: SRX2692198 (N. ampullaria), SRX2692197 (N. rafflesiana), and SRX2692196 (N. × hookeriana) (Zulkapli et al., 2017). Consensus isoform sequences can be accessed from the TSA repository: {"type":"entrez-nucleotide","attrs":{"text":"GGLJ00000000.1","term_id":"1394787833"}}GGLJ00000000.1 (N. ampullaria), {"type":"entrez-nucleotide","attrs":{"text":"GGLG00000000.1","term_id":"1394787835"}}GGLG00000000.1 (N. rafflesiana), and {"type":"entrez-nucleotide","attrs":{"text":"GGLF00000000.1","term_id":"1394787834"}}GGLF00000000.1 (N. × hookeriana).