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Human Gut Microbiota–Transplanted Gn Pig Models for HRV Infection
Published in Lijuan Yuan, Vaccine Efficacy Evaluation, 2022
Sequencing reads were processed with Quantitative Insights into Microbial Ecology (QIIME) (Caporaso et al., 2010). High-quality reads with Phred quality score ≥20 (corresponding to a sequencing error rate ≤0.01) were clustered into operational taxonomic units (OTUs) with the program UCLUST (Edgar, 2010). Chimeric sequences were identified with CHIMERASLAYER (Haas et al., 2011) and removed from further analysis. Bacterial taxonomy was assigned by using a naïve Bayes classifier (Wang et al., 2007b) against reference databases and bacterial taxonomy maps at Greengenes (McDonald et al., 2012). A phylogenetic tree was constructed (Price et al., 2010) from PyNAST-aligned sequences representing each OTU. Principle coordinate analysis on stool samples was based on UniFrac distances (Lozupone and Knight, 2005). Distance-based redundancy analysis for the effect of HRV on community structures was performed with the Vegan package (Vegan: Community Ecology Package, 2013). Shannon and Simpson diversity indices and a rank abundance curve were both generated with QIIME.
Determining the accuracy of next generation sequencing based copy number variation analysis in Hereditary Breast and Ovarian Cancer
Published in Expert Review of Molecular Diagnostics, 2022
Nihat Bugra Agaoglu, Busra Unal, Ozlem Akgun Dogan, Payam Zolfagharian, Pari Sharifli, Aylin Karakurt, Burak Can Senay, Tugba Kizilboga, Jale Yildiz, Gizem Dinler Doganay, Levent Doganay
The NGS raw data generated by Illumina MiSeq and NextSeq 500 is in FASTQ format, which contains quality scores of each base. All samples were analyzed in a single workflow for SNV, INDEL, and CNVs with the Sophia Genetics Data Driven Medicine (DDM) platform. Fastq DNA sequence files, with a Phred Quality Score of 30 (Q30), were automatically uploaded and immediately processed by specific algorithms and machine learning approaches. The sequences were mapped to the hg19 human reference genome, and CNV regions were then evaluated by the Sophia Genetics MUSKAT algorithm. The CNVs were identified by measuring the coverage levels of the desired regions along with samples in the same run. The CNV attributions were also classified by high or medium confidence level, high being less than 50 mapped reads, and samples that do not achieve this quality level are considered as rejected analysis.
Molecular regulation of adhesion and biofilm formation in high and low biofilm producers of Bacillus licheniformis using RNA-Seq
Published in Biofouling, 2019
Faizan Ahmed Sadiq, Steve Flint, Hafiz Arbab Sakandar, GuoQing He
The details regarding raw data including the Phred quality score (Q20 and Q30) are given in Table S1 (Supplemental material is available online). An average of 9,498,144 (planktonic phenotype) and 8,339,212 (biofilm phenotype) sequencing reads for the strain H and an average total of 9,414,898 (planktonic phenotype) and 9,060,116 (biofilm phenotype) clean reads for the strain L were obtained from the cDNA libraries after quality trimming and removal of duplicates (Table 1). Out of the total reads sequenced per sample, ∼80% mapped to the reference B. licheniformis (ATCC 14580) genome. The detailed information regarding the total reads and the percentage which mapped on the reference genome are given in Table 1. An average of 3,746 and 3,634 genes were detected in H-biofilm and H-planktonic samples, respectively. Similarly, 3,815 and 3,630 genes were detected in L-biofilm and L-planktonic samples, respectively (Figure 1a).
Diversity and functional analysis of salivary microflora of Indian Antarctic expeditionaries
Published in Journal of Oral Microbiology, 2019
Brij Bhushan, A. P. Yadav, S. B. Singh, L. Ganju
The 16S analysis was performed using CLC microbial Genomics Module v2.0 (Qiagen, Valencia, CA). Quality filtering consisted of discarding reads <200 bp and >1,000 bp, excluding homopolymer runs >6 bp and ambiguous bases >6 bp, accepting one barcode correction and two primer mismatches. A value of 25 mer was considered as the minimum average. Phred quality score allowed in reads in a sliding window of 50 bp followed by hos sequences removed (Homosapiens hg 19). Paired reads were merged using the Optional Merge Paired Reads tool at default parameters-mismatch: 2, mismatch score: Default 8 (an overlap of 11 bases with one mismatch), gap cost: 2 (an insertion or deletion in the alignment). For clustering the merged sequences, all reads were trimmed to the same length using the fixed length-trimming algorithm and calculated as the mean length of the merged reads minus one standard deviation for the combined reads in all samples [24]. Finally, the operational taxonomic units (OTUs) clustering tool clustered fixed length trimmed reads to OTUs at 97% similarity. Chimeras were removed, generating abundance of the OTUs for all samples (default abundance 10), with Singleton OTUs removed for statistical analysis. The analysis tool used for taxonomy assignment was performed employing the naïve Bayesian RDP classifier with a minimum confidence of 0.8 against the Green genes database resulting in the names of the identified taxa (assemblies).