Primer pairs (Online resource 5) for quantitative RT-PCR (RT-qPCR) were designed as recommended in (Citri et al. 2012 ) using the CLC main workbench primer design software tool (CLC Main Workbench Version 8.0.1, QIAGEN www.qiagenbioinformatics.com ). PCR amplification efficiencies of the primer pairs for the 91 targets and 2 endogenous control genes were validated as suggested by Schmittgen and Livak (2008 (link)). Standard RT-qPCRs were performed using the Power SYBR-Green PCR Master Mix (Applied Biosystems). The specificity of the amplification was affirmed by visual inspection of the amplification products followed by melting curve analysis and gel electrophoresis of the PCR products (after 40 thermal cycles, size inspection on 3% agarose gels).
Clc main workbench version 8
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Sourced in Germany
CLC Main Workbench version 8.0 is a bioinformatics software suite developed by Qiagen. It provides a range of tools for the analysis and visualization of biological data, including DNA, RNA, and protein sequences.
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Lab products found in correlation
8 protocols using clc main workbench version 8
1
Quantitative RT-PCR Primer Design and Validation
2
Quantitative RT-qPCR Primer Design and Validation
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Primers (listed in Online resource 3) were designed as recommended in (Citri et al. 2012 ) using the CLC-main workbench primer design software tool (CLC Main Workbench Version 8.0.1, QIAGEN www.qiagenbioinformatics.com). Standard RT-qPCRs were performed using the PowerSYBR-Green PCR Master Mix (Applied Biosystems). The specificity of the amplicons was assessed by visual inspection of the amplification and melting curves of the RT-qPCR and by gel electrophoresis of the PCR products (after 40 thermal cycles with size inspection on 3% agarose). PCR amplification efficiencies of the primer pairs for the 91 target genes and two endogenous control genes were validated as suggested in step 14 and 15 of the protocol of Schmittgen and Livak (2008) .
3
Multiplex rtPCR Assays for Respiratory and Enteric Pathogens
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Eleven rtPCR assays targeting respiratory and enteric viral and bacterial pathogens were established (Table 1 ). The primer and probe sequences were copied either from previously published assays or designed in this study. Some of the published primer and probe sequences were modified to improve the specificity or to adapt to the selected PCR conditions. New primer and probe sequences were designed based on alignments containing sequences of the target gene for the selected pathogens. The sequences were retrieved from GenBank (35 (link)) and aligned using CLC Main Workbench version 8.0 (QIAGEN). The specificity of the oligonucleotides were tested in silico using nucleotide BLAST search (36 (link)), and the melting temperature and basic properties were approximated using OligoCalc (37 (link)). The oligonucleotides were purchased from Eurofins Genomics (Ebersberg, Germany).
4
Bioinformatic Analysis of Tp9 Sequences
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Raw nucleotide sequences were confirmed to be Tp9 sequences by sequence similarity analysis using the Basic Local Alignment Search Tool (BLAST) executed on the National Centre for Biotechnology Information (NCBI) platform. Sequence assembly, editing and translation was done using CLC Main Workbench version 8.0 (Qiagen, Germany). Multiple sequence alignment of consensus sequences together with the Tp9 reference sequence (Table 4) was done using the online version 7 of MAFFT (Katoh and Standley, 2013) applying the default parameters (http://mafft.cbrc.jp/alignment/server/). Aligned sequences/sequence matrices were viewed, edited manually and truncated using MEGA version 7 (Kumar et al., 2016) . Phylogenetic analysis of predicted protein sequences selected based on the epitope variants was performed using MEGA version 7 (Kumar et al., 2016) . Evolutionary history was inferred using Maximum Likelihood, by applying the default substitution model (General Reverse Transcriptase model) in MEGA 7, and the phylogeny tested using 100 bootstrap replicates, where a bootstrap support ≥0.7 (70%) was considered significant.
5
Bioinformatic Analysis of p67 Sequences
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Raw p67 sequences were confirmed using the Basic Local Alignment Search Tool (BLAST), and sequence assembly and editing were done using the CLC Main Workbench version 8.0 (Qiagen, Hilden, Germany). Multiple sequence alignment of consensus sequences, together with reference sequences (S6 Table ), was done using the online version 7 of MAFFT [38 (link)] applying the default parameters (http://mafft.cbrc.jp/alignment/server/ ). Estimation of the effect of amino acid substitutions within the epitope regions was done using SIFT predictions [39 (link)] where a probability score ˃0.05 was predicted to be tolerant.
6
Sequence Alignment and Variant Analysis
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Raw p67 sequences were confirmed using the Basic Local Alignment Search Tool (BLAST), and sequence assembly and editing were done using the CLC Main Workbench version 8.0 (Qiagen, Hilden, Germany). Multiple sequence alignment of consensus sequences, together with reference sequences (Supp. Table S6), was done using the online version 7 of MAFFT (37) applying the default parameters (http://mafft.cbrc.jp/alignment/server/). Estimation of the effect of amino acid substitutions within the epitope regions was done using SIFT predictions (38) where a probability score ˃0.05 was predicted to be tolerant.
7
Phylogenetic Analysis Using CLC and PhyML
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Sequence alignments and tree constructions were performed using CLC Main Workbench version 8.1 (QIAGEN, Aarhus) and PhyML version 3.3.20190321 (Guindon et al. 2009 (link)).
8
Visualizing Pseudo-time Dynamics in Protein Structures
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First, the median value pseudo-time projection generated from sc-Tiling was summarized for each sgRNA. To depict the pseudo-time score over regions with no sgRNA coverage, we interpolated the signal via Gaussian kernel smoothing in R38 (link). The bandwidth was defined by the maximum gap length of the non-covered regions for local smoothing due to regional uneven sgRNA densities. To map the smoothed pseudo-time score to peptide positions, the average pseudo-time score over the trinucleotide codons was calculated for each peptide position. Pairwise alignments of primary amino acid sequences were performed using CLC Main Workbench version 8.1 (Qiagen) to ensure functional annotations of the smoothed pseudo-time scores of mouse Dot1l sc-Tiling data onto human DOT1L protein structures. Atomic data of macromolecular structures were retrieved from the Research Collaboratory for Structural Bioinformatics Protein Data Bank (RCSB Protein Data Bank (PDB) at https://www.rcsb.org )39 (link) in PDB file format. The PDB files were visualized and analyzed using UCSF Chimera (version 1.14 build 42000)40 (link). Subsequently, the smoothed pseudo-time scores were mapped onto three-dimensional protein structures using the “Defined Attribute” and “Render by Attribute” functionalities in UCSF Chimera40 (link).
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