Sequel iie system

Genomic DNA was subjected to HiFi SMRTbell library construction using the SMRTbell Express Template Prep Kit 2.0 (PacBio), according to the manufacturer’s instructions, with a minor modification. Because the genomic DNA was degraded, the DNA shearing step recommended in the protocol was skipped. The resultant DNA was fractionated with BluePippin (Sage Science) to eliminate fragments less than 10 kb in size. The DNA libraries prepared from the two sporocarps were indexed with unique barcode adapters, and sequenced on a single SMRT cell 8M on the Sequel IIe system (PacBio).

Leaf tissue was sampled from edited line 19Q4-13 and wild type Tx430 sorghum and high molecular weight DNA was extracted using Circulomics Nanobind Plant Nuclei Big DNA Kit. HiFi libraries were generated using PacBio SMRTbell Express Template Prep Kit 2.0. The libraries were sequenced on a PacBio Sequel-IIe system in HiFi-CCS mode.
Read alignment and variant calling was performed using a local assembly approach. Reads were aligned using PBMM2, NGMLR and lra. Reads which aligned to the alpha kafirin region in any alignment method were extracted and assembled into contigs using canu. The assembled local contigs were aligned to the Tx430 reference genome using lra on CONTIG mode. The contigs spanning the kafirin region was used in conjunction with MuMMER to characterize the edits induced by CRISPR/Cas9.

Fecal samples were processed and analyzed as previously described^{16 (link)}. Briefly, fecal DNA was purified, PCR amplified, and pooled for sequencing using the Complete StrainID Kit (StrainID set A [barcodes 1 to 96]; Intus Biosciences, Farmington, CT) according to the manufacturer’s instructions. Amplicon libraries were created using the SMRTbell express template prep kit 2.0 (catalog number 100-938-900; PacBio). The library was sequenced on a Sequel IIe system (Pacific Biosciences) at the University of Delaware, Delaware Biotechnology Institute Sequencing and Genotyping Center, Newark, DE. The selected reads from each sample were primer trimmed and filtered to reads within the length range of 1900–3000 bp. The trimmed and filtered reads were analyzed manually via a histogram to identify peaks of read lengths that are likely to represent unique amplicons. The corresponding read length ranges (i.e., the 2400- to 2405-bp range from each sample) were passed to DADA2^{45 (link)} and pooled for ASV inference^{16 (link)}. A sequence table of ASV abundance per sample was produced as part of the DADA2 output, and a heat map was generated in R using the sequence table. SBanalyzer 2.4 (Intus Biosciences) was used to map ccs reads to the Athena database and assign taxonomic identification^{16 (link)}.