Pacbio sequencing

Genomic DNA was sequenced with Nanopore sequencers (Oxford Nanopore Technologies, UK). The genome structure of HIS019 and HIS471 was confirmed with PacBio sequencing (Pacific Biosciences, USA). For long-read sequencing, genomic DNA (6 mg) was treated with a Short-Read Eliminator Kit XS (Circulomics) to remove fragments < 10 kbp, and libraries were prepared using a Rapid Barcoding Sequencing Kit (SQK-RBK004, Oxford Nanopore Technologies). Sequencing was performed on the MinION (Sample HIS002 and HIS019) and GridION X5 (Sample HIS631, HIS641, HIS016, HIS471) systems using eight R9.4 flow cells. PacBio library construction and sequencing with Sequel II (Pacific Biosciences, USA) was outsourced (Takara Bio, Japan). Illumina paired-end genomic libraries with insert sizes of 300–350 bp were constructed with a Nextera DNA Flex Library Prep Kit (Illumina, USA). The libraries were sequenced with the NextSeq 500/550 Mid Output Kit v2.5 (Illumina, USA) for 151 bp from both ends. Illumina RNA-seq libraries were constructed with the NEBNext Ultra II Directional RNA Library Prep Kit (New England Biolabs, USA) for Illumina and sequenced with the NextSeq 500/550 Mid Output Kit v2.5 (Illumina, USA) for 151 bp from both ends.

DNA from a sample with high proportional abundance of TM7-H1 was sent to Pacific Biosciences for PacBio sequencing, using the TdT protocol, which is suitable for sequencing low-input samples. An HGAP metagenome assembly was performed using a white list to exclude reads mapped to human, which yielded three TM7-H1 contigs. Genome sequences were annotated with in-house pipelines using Prokka and ASGARD^{66 (link)}.

Genomic DNA was sequenced with Nanopore sequencers (Oxford Nanopore Technologies, UK). The genome structure of HIS019 and HIS471 was confirmed with PacBio sequencing (Pacific Biosciences, USA). For long-read sequencing, genomic DNA (6 mg) was treated with a Short-Read Eliminator Kit XS (Circulomics) to remove fragments < 10 kbp, and libraries were prepared using a Rapid Barcoding Sequencing Kit (SQK-RBK004, Oxford Nanopore Technologies). Sequencing was performed on the MinION (Sample HIS002 and HIS019) and GridION X5 (Sample HIS631, HIS641, HIS016, HIS471) systems using eight R9.4 flow cells. PacBio library construction and sequencing with Sequel II (Pacific Biosciences, USA) was outsourced (Takara Bio, Japan). Illumina paired-end genomic libraries with insert sizes of 300–350 bp were constructed with a Nextera DNA Flex Library Prep Kit (Illumina, USA). The libraries were sequenced with the NextSeq 500/550 Mid Output Kit v2.5 (Illumina, USA) for 151 bp from both ends. Illumina RNA-seq libraries were constructed with the NEBNext Ultra II Directional RNA Library Prep Kit (New England Biolabs, USA) for Illumina and sequenced with the NextSeq 500/550 Mid Output Kit v2.5 (Illumina, USA) for 151 bp from both ends.

Bacterial community diversity was analysed via single‐molecule real‐time PacBio sequencing (Pacific Biosciences, Menlo Park, CA).³⁴, ³⁵ The full‐length 16S ribosomal RNA gene was amplified from genomic DNA using the bacteria‐specific primers 27 F (5′‐AGAGTTTGATCMTGGCTCAG) and 1492 R (5′‐TACGGYTACCTTGTTACGACTT). The quality and concentration of the PCR products were determined via electrophoresis on 2% agarose gels. Sequencing libraries were constructed using the SMRTbell Template Prep Kit 1.0‐SPv3 according to the manufacturer's instructions (Pacific Biosciences).
No sequences contained ambiguous bases. Details of the sequencing output, such as raw reads, clean reads and average read lengths, are summarized in supplemental Table S1. Operational taxonomic unit (OTU) clustering was performed using a 99% similarity cut‐off to represent one species per OTU. After sequencing data were rarefied, the alpha and beta diversities were calculated, and differences in relative abundance were compared using Statistical Analyses of Metagenomic Profiles (STAMP) software.

Genomic DNA was isolated from peripheral red blood cells using standard method of Proteinase K-SDS digestion, ammonium acetate protein precipitation, and precipitation of nucleic acids by 2-propanol. High molecular weight (HMW) DNA was randomly sheared to produce a 350-bp insert library, and paired-end sequences were produced on an Illumina NextSeq 500 platform. For the blue catfish long reads, HMW DNA was sheared with a Covaris® G-tube targeting > 20 kb fragments. Sheared DNA was prepared for PacBio sequencing (Pacific Biosciences, Menlo Park CA) using the SMRTbell™ Template Prep Kit, and size selected with the Blue Pippin (Sage Sciences). Sequencing was performed on a PacBio® RS II System on SMRT®Cell 8Pac V3 cells using P6-C4 chemistry. To target Continuous Long Reads, the libraries were sequenced using 6-h movies on 90 SMRT®Cells. For the channel catfish long reads, HMW libraries were produced as described for blue catfish above and sequencing was performed on a PacBio® Sequel System on 12 LR SMRT®Cells 1 M v3 using SMRTLink version 6 software. Continuous Long Reads were produced using 15-h movies. This channel catfish sample was also run on an 8 M SMRT®Cell on a PacBio® Sequel II System using v7.0 software.

Each gene was targeted with oligonucleotide baits (Agilent Technologies, Santa Clara, CA; Roche, Pleasanton, CA; IDT, Coralville, IA) to capture exons, the 10 to 20 bases flanking intronic sequences, and noncoding regions of clinical interest. Baits were iteratively balanced to obtain a minimum of 50X and an average of 350X depth-of-sequence read coverage across all targeted areas. Sequencing was performed on HiSeq and NovaSeq instruments (Illumina, San Diego, CA). A suite of bioinformatics methods was used to identify single nucleotide variants, small and large insertions/deletions, exon-level deletions and duplications, and rare structural or mosaic variants [20 , 21 ].
Genomic DNA extracted from patient blood or saliva was processed by next-generation sequencing as described previously [20 ]. Variants requiring confirmation were confirmed using an orthogonal method, such as PacBio sequencing (Pacific Biosciences, Menlo Park, CA) or exon-focused microarray-based comparative genomic hybridization (Agilent Technologies, Santa Clara, CA) [21 ]. Clinically reported variants and de-identified clinical information, if provided, were collected for analyses.