Software package

Primers for conventional PCR detection of PCV2, PCV1, PPV, PRRSV, CSFV and PRV were present in Table 2. Primers and probes for UNDP-PCR detection system were designed by comparing multiple sequences of PCV2a- and PCV2b-encoding gene using DNASTAR software package (DNASTAR, inc., Wisconsin, USA). The highly conserved sequences in the capsid protein-coding region of different PCV2a and PCV2b strains were selected for designing primers and probes (Table 2). The designed primers and probes have higher specificity to ensure precise diagnosis. The primers and probes were synthesized by sangon (Shanghai, China).

The activated sludge at day 96 in the reactors was chosen to construct the SDIMO and CYP153 gene clone libraries. PCR products were cloned using the pMD19-T vector (TaKaRa, Bio Inc., Shiga, Japan) according to the manufacturer's instructions. At least 20 positive clones for SDIMO genes and 60 positive clones for CYP153 genes from each sample were selected for sequencing (Sangon Biotech, Shanghai, China). The sequences were edited using the DNAstar software package (DNAstar, USA). The sequences were confirmed online by NCBI BLAST and aligned using ClustalX (Version 1.81). The diversity was determined by rarefaction analysis using PHYLIP (Version 3.69). The sequences displaying more than 97% identity with each other were grouped into one operational taxonomic unit (OTU) by using the furthest neighbor algorithm in the DOTUR program (Schloss and Handelsman, 2005 (link)). The coverage of the clone libraries was calculated as previously described (Hill et al., 2003 (link)). All OTU representative sequences, their nearest neighbors and some reference sequences were analyzed by BLAST and imported in MEGA (Version 5) to construct unrooted phylogenetic trees using the neighbor-joining method. The relative confidence of the tree topologies was evaluated by performing 1000 bootstrap replicates.

The Genbank accession number for TP901-1 is NC_002747. The sequence of TMP_TP901-1, corresponding to the product of ORF45, was manipulated using DNASTAR software package (Version 7.2, 2007; DNASTAR Inc., Madison, WI). Repeat sequences in TMP_TP901-1 were deduced by manually aligning Trp and Phe residues with 11 or 18 amino acid periodicity. Hydrophobic and predicted transmembrane domains were identified using TMHMM35 (link), while protein transmembrane topology was characterised using TOPO236 (link). The secondary structure of TMP_TP901-1 was predicted using SABLE37 (link) and alignment of TMP amino acid sequences was performed using ClustalW38 (link).

Sanger sequencing was performed to confirm and segregate the obtained results through whole-exome sequencing and to screen the TUBA3D gene in 200 unrelated sporadic KC patients. Primers were designed to amplify all five coding fragments and the intron–exon boundaries of the TUBA3D gene (NM_080386.3, see Supplementary Table S1). Every target fragment was amplified using Taq DNA polymerase (Takara, Dalian, China). The products were purified with alkaline phosphatase (Shrimp) (Takara, Dalian, China) and exonuclease I (Takara, Dalian, China), and subjected to direct DNA sequencing using the BigDye™ Terminator v3.1 Cycle Sequencing kit and ABI PRISM 3730 sequencer (Applied Biosystems Inc., USA). Sequences were aligned and analyzed using the DNASTAR software package (DNASTAR Inc., USA).
The novel mutations of TUBA3D were also genotyped in 200 unrelated healthy controls using high-resolution melt (HRM) analysis or Sanger sequencing. The primers used for genotyping the mutations of TUBA3D are shown in Supplementary Table S2. For HRM, the amplification assays were performed using the Type-it HRM PCR Kit (QIAGEN, Germany) on the Rotor-Gene Q Real-Time PCR system (QIAGEN, Germany). The results were obtained and analyzed using the Rotor-Gene Q Series Software (version 2.1.0). The Sanger sequencing method is the same as the above description.