Hybridization:Article Title: Prospective metagenomic sequencing of wastewater across the United States yields robust viral enrichment and concordance with digital PCR measurements
Article Snippet: .. Prepared libraries were enriched using the Twist Comprehensive Viral Research Panel (Twist Biosciences, #103548), which, as reported by the manufacturer, targets more than 3,000 viral genomes through hybridization at 70°C for 16 h. Bacterial, human, and other mammalian ribosomal RNA (rRNA) was depleted from the samples using CRISPR-Cas9 technology via the CRISPRclean Stranded Total RNA Prep with rRNA Depletion kit (Jumpcode Genomics, San Diego, CA #KIT1014). .. Prepared libraries were sequenced on an Illumina (Foster City, CA) NovaSeq X Plus (10B flow cells, 150-bp paired-end reads), alongside negative sequencing controls.
Article Title: Four Years of Continuous Weekly Wastewater Analysis Reveals Sequencing as a Robust Alternative to PCR for Respiratory Virome Surveillance
Article Snippet: .. Probe hybridization was carried out with Twist Comprehensive Viral Research panel (TWIST Biosciences) at 70°C for 16 hrs. .. Post-capture pools were further amplified with 12 PCR cycles, and final libraries were sequenced on the Illumina NovaSeq 6000 SP flow cell to generate 2×150 bp paired-end sequence reads.
CRISPR:Article Title: Prospective metagenomic sequencing of wastewater across the United States yields robust viral enrichment and concordance with digital PCR measurements
Article Snippet: .. Prepared libraries were enriched using the Twist Comprehensive Viral Research Panel (Twist Biosciences, #103548), which, as reported by the manufacturer, targets more than 3,000 viral genomes through hybridization at 70°C for 16 h. Bacterial, human, and other mammalian ribosomal RNA (rRNA) was depleted from the samples using CRISPR-Cas9 technology via the CRISPRclean Stranded Total RNA Prep with rRNA Depletion kit (Jumpcode Genomics, San Diego, CA #KIT1014). .. Prepared libraries were sequenced on an Illumina (Foster City, CA) NovaSeq X Plus (10B flow cells, 150-bp paired-end reads), alongside negative sequencing controls.
Sequencing:Article Title: Four Years of Continuous Weekly Wastewater Analysis Reveals Sequencing as a Robust Alternative to PCR for Respiratory Virome Surveillance
Article Snippet: .. Probe panel-based hybrid-capture sequencing, for instance, can be used to detect a wide array of microbial targets within the same sample (3,153 viral targets with TWIST Bioscience’s Comprehensive Viral Research Panel ). ..
Article Title: Sequencing the wastewater virome: Current strategies and bioinformatic pitfalls.
Article Snippet: .. Continued Study Target(s) Platform Pipeline/database Read count and depth/coverage (where reported) Key outcome Main limitations/ comments Probe-based targeted sequencing Karatas et al.42 TWIST Comprehensive Viral Panel AVITI EsViritu; Virus Pathogen Database > 3 million total reads mapped to viruses (n = 6; visual estimates from plots); reads were mapped with 90% percentage coverage criteria 2294 viral genomes or segments recovered, spanning 51 animal- and 135 human-associated viral species detection is biased toward known or closely related viruses (probe efficiency limited to sequences with > 80% nt similarity); limited assessment of viral population complexity (analysis focused on consensus genomes) Tisza et al.20 TWIST Comprehensive Viral Panel Illumina NovaSeq 6000 EsViritu; Virus Pathogen Database median 28.5 genomes/segments covered over > 90% of their length, additional 41 covered over > 50% of their length (percentage coverage; n = 363) broad community viral diversity: 465 virus strains detected with reads correlating with clinical data for some insufficient genome coverage for reliable allele/variant frequency analysis; low viral shedding limits coverage; Improved enrichment and increased depth are required to enhance coverage and variant analysis Zulli et al.34 Illumina Viral Surveillance Panel Illumina NovaSeq 6000 Chan-Zuckerberg Infectious Disease mNGS Illumina; minimap2; SPADES; bowtie2; NCBI NT/ NR databases ∼40 million reads per sample; 3%–90% percentage coverage, 5- to 20-fold read depth 33 respiratory panel viruses detected, enabling seasonal monitoring of outbreak timing and severity (including viruses with limited clinical surveillance) practical constraints necessitated sample pooling; varying coverage breadth and depth among targets (influenced by viral concentration/ outbreak activity); non-customized bioinformatics workflow limits analysis performance; matrix complexity affects sequencing performance (Continued on next page) 6 iS c ie n c e 2 9 , 1 1 7 5 9 6 , O c to b e r 1 6 , 2 0 2 6 iS cience R e v ie w ll O P E N A C C E S S iS c ie n c e 2 9 , 1 1 7 5 9 6 , O c to b e r 1 6 , 2 0 2 6 7 iS cience R e v ie w adapter ligation to the DNA template (Figure 2). ..
Targeted Sequencing:Article Title: Sequencing the wastewater virome: Current strategies and bioinformatic pitfalls.
Article Snippet: .. Continued Study Target(s) Platform Pipeline/database Read count and depth/coverage (where reported) Key outcome Main limitations/ comments Probe-based targeted sequencing Karatas et al.42 TWIST Comprehensive Viral Panel AVITI EsViritu; Virus Pathogen Database > 3 million total reads mapped to viruses (n = 6; visual estimates from plots); reads were mapped with 90% percentage coverage criteria 2294 viral genomes or segments recovered, spanning 51 animal- and 135 human-associated viral species detection is biased toward known or closely related viruses (probe efficiency limited to sequences with > 80% nt similarity); limited assessment of viral population complexity (analysis focused on consensus genomes) Tisza et al.20 TWIST Comprehensive Viral Panel Illumina NovaSeq 6000 EsViritu; Virus Pathogen Database median 28.5 genomes/segments covered over > 90% of their length, additional 41 covered over > 50% of their length (percentage coverage; n = 363) broad community viral diversity: 465 virus strains detected with reads correlating with clinical data for some insufficient genome coverage for reliable allele/variant frequency analysis; low viral shedding limits coverage; Improved enrichment and increased depth are required to enhance coverage and variant analysis Zulli et al.34 Illumina Viral Surveillance Panel Illumina NovaSeq 6000 Chan-Zuckerberg Infectious Disease mNGS Illumina; minimap2; SPADES; bowtie2; NCBI NT/ NR databases ∼40 million reads per sample; 3%–90% percentage coverage, 5- to 20-fold read depth 33 respiratory panel viruses detected, enabling seasonal monitoring of outbreak timing and severity (including viruses with limited clinical surveillance) practical constraints necessitated sample pooling; varying coverage breadth and depth among targets (influenced by viral concentration/ outbreak activity); non-customized bioinformatics workflow limits analysis performance; matrix complexity affects sequencing performance (Continued on next page) 6 iS c ie n c e 2 9 , 1 1 7 5 9 6 , O c to b e r 1 6 , 2 0 2 6 iS cience R e v ie w ll O P E N A C C E S S iS c ie n c e 2 9 , 1 1 7 5 9 6 , O c to b e r 1 6 , 2 0 2 6 7 iS cience R e v ie w adapter ligation to the DNA template (Figure 2). ..
Virus:Article Title: Sequencing the wastewater virome: Current strategies and bioinformatic pitfalls.
Article Snippet: .. Continued Study Target(s) Platform Pipeline/database Read count and depth/coverage (where reported) Key outcome Main limitations/ comments Probe-based targeted sequencing Karatas et al.42 TWIST Comprehensive Viral Panel AVITI EsViritu; Virus Pathogen Database > 3 million total reads mapped to viruses (n = 6; visual estimates from plots); reads were mapped with 90% percentage coverage criteria 2294 viral genomes or segments recovered, spanning 51 animal- and 135 human-associated viral species detection is biased toward known or closely related viruses (probe efficiency limited to sequences with > 80% nt similarity); limited assessment of viral population complexity (analysis focused on consensus genomes) Tisza et al.20 TWIST Comprehensive Viral Panel Illumina NovaSeq 6000 EsViritu; Virus Pathogen Database median 28.5 genomes/segments covered over > 90% of their length, additional 41 covered over > 50% of their length (percentage coverage; n = 363) broad community viral diversity: 465 virus strains detected with reads correlating with clinical data for some insufficient genome coverage for reliable allele/variant frequency analysis; low viral shedding limits coverage; Improved enrichment and increased depth are required to enhance coverage and variant analysis Zulli et al.34 Illumina Viral Surveillance Panel Illumina NovaSeq 6000 Chan-Zuckerberg Infectious Disease mNGS Illumina; minimap2; SPADES; bowtie2; NCBI NT/ NR databases ∼40 million reads per sample; 3%–90% percentage coverage, 5- to 20-fold read depth 33 respiratory panel viruses detected, enabling seasonal monitoring of outbreak timing and severity (including viruses with limited clinical surveillance) practical constraints necessitated sample pooling; varying coverage breadth and depth among targets (influenced by viral concentration/ outbreak activity); non-customized bioinformatics workflow limits analysis performance; matrix complexity affects sequencing performance (Continued on next page) 6 iS c ie n c e 2 9 , 1 1 7 5 9 6 , O c to b e r 1 6 , 2 0 2 6 iS cience R e v ie w ll O P E N A C C E S S iS c ie n c e 2 9 , 1 1 7 5 9 6 , O c to b e r 1 6 , 2 0 2 6 7 iS cience R e v ie w adapter ligation to the DNA template (Figure 2). ..
Analysis:Article Title: Sequencing the wastewater virome: Current strategies and bioinformatic pitfalls.
Article Snippet: .. Continued Study Target(s) Platform Pipeline/database Read count and depth/coverage (where reported) Key outcome Main limitations/ comments Probe-based targeted sequencing Karatas et al.42 TWIST Comprehensive Viral Panel AVITI EsViritu; Virus Pathogen Database > 3 million total reads mapped to viruses (n = 6; visual estimates from plots); reads were mapped with 90% percentage coverage criteria 2294 viral genomes or segments recovered, spanning 51 animal- and 135 human-associated viral species detection is biased toward known or closely related viruses (probe efficiency limited to sequences with > 80% nt similarity); limited assessment of viral population complexity (analysis focused on consensus genomes) Tisza et al.20 TWIST Comprehensive Viral Panel Illumina NovaSeq 6000 EsViritu; Virus Pathogen Database median 28.5 genomes/segments covered over > 90% of their length, additional 41 covered over > 50% of their length (percentage coverage; n = 363) broad community viral diversity: 465 virus strains detected with reads correlating with clinical data for some insufficient genome coverage for reliable allele/variant frequency analysis; low viral shedding limits coverage; Improved enrichment and increased depth are required to enhance coverage and variant analysis Zulli et al.34 Illumina Viral Surveillance Panel Illumina NovaSeq 6000 Chan-Zuckerberg Infectious Disease mNGS Illumina; minimap2; SPADES; bowtie2; NCBI NT/ NR databases ∼40 million reads per sample; 3%–90% percentage coverage, 5- to 20-fold read depth 33 respiratory panel viruses detected, enabling seasonal monitoring of outbreak timing and severity (including viruses with limited clinical surveillance) practical constraints necessitated sample pooling; varying coverage breadth and depth among targets (influenced by viral concentration/ outbreak activity); non-customized bioinformatics workflow limits analysis performance; matrix complexity affects sequencing performance (Continued on next page) 6 iS c ie n c e 2 9 , 1 1 7 5 9 6 , O c to b e r 1 6 , 2 0 2 6 iS cience R e v ie w ll O P E N A C C E S S iS c ie n c e 2 9 , 1 1 7 5 9 6 , O c to b e r 1 6 , 2 0 2 6 7 iS cience R e v ie w adapter ligation to the DNA template (Figure 2). ..
Variant Assay:Article Title: Sequencing the wastewater virome: Current strategies and bioinformatic pitfalls.
Article Snippet: .. Continued Study Target(s) Platform Pipeline/database Read count and depth/coverage (where reported) Key outcome Main limitations/ comments Probe-based targeted sequencing Karatas et al.42 TWIST Comprehensive Viral Panel AVITI EsViritu; Virus Pathogen Database > 3 million total reads mapped to viruses (n = 6; visual estimates from plots); reads were mapped with 90% percentage coverage criteria 2294 viral genomes or segments recovered, spanning 51 animal- and 135 human-associated viral species detection is biased toward known or closely related viruses (probe efficiency limited to sequences with > 80% nt similarity); limited assessment of viral population complexity (analysis focused on consensus genomes) Tisza et al.20 TWIST Comprehensive Viral Panel Illumina NovaSeq 6000 EsViritu; Virus Pathogen Database median 28.5 genomes/segments covered over > 90% of their length, additional 41 covered over > 50% of their length (percentage coverage; n = 363) broad community viral diversity: 465 virus strains detected with reads correlating with clinical data for some insufficient genome coverage for reliable allele/variant frequency analysis; low viral shedding limits coverage; Improved enrichment and increased depth are required to enhance coverage and variant analysis Zulli et al.34 Illumina Viral Surveillance Panel Illumina NovaSeq 6000 Chan-Zuckerberg Infectious Disease mNGS Illumina; minimap2; SPADES; bowtie2; NCBI NT/ NR databases ∼40 million reads per sample; 3%–90% percentage coverage, 5- to 20-fold read depth 33 respiratory panel viruses detected, enabling seasonal monitoring of outbreak timing and severity (including viruses with limited clinical surveillance) practical constraints necessitated sample pooling; varying coverage breadth and depth among targets (influenced by viral concentration/ outbreak activity); non-customized bioinformatics workflow limits analysis performance; matrix complexity affects sequencing performance (Continued on next page) 6 iS c ie n c e 2 9 , 1 1 7 5 9 6 , O c to b e r 1 6 , 2 0 2 6 iS cience R e v ie w ll O P E N A C C E S S iS c ie n c e 2 9 , 1 1 7 5 9 6 , O c to b e r 1 6 , 2 0 2 6 7 iS cience R e v ie w adapter ligation to the DNA template (Figure 2). ..
Concentration Assay:Article Title: Sequencing the wastewater virome: Current strategies and bioinformatic pitfalls.
Article Snippet: .. Continued Study Target(s) Platform Pipeline/database Read count and depth/coverage (where reported) Key outcome Main limitations/ comments Probe-based targeted sequencing Karatas et al.42 TWIST Comprehensive Viral Panel AVITI EsViritu; Virus Pathogen Database > 3 million total reads mapped to viruses (n = 6; visual estimates from plots); reads were mapped with 90% percentage coverage criteria 2294 viral genomes or segments recovered, spanning 51 animal- and 135 human-associated viral species detection is biased toward known or closely related viruses (probe efficiency limited to sequences with > 80% nt similarity); limited assessment of viral population complexity (analysis focused on consensus genomes) Tisza et al.20 TWIST Comprehensive Viral Panel Illumina NovaSeq 6000 EsViritu; Virus Pathogen Database median 28.5 genomes/segments covered over > 90% of their length, additional 41 covered over > 50% of their length (percentage coverage; n = 363) broad community viral diversity: 465 virus strains detected with reads correlating with clinical data for some insufficient genome coverage for reliable allele/variant frequency analysis; low viral shedding limits coverage; Improved enrichment and increased depth are required to enhance coverage and variant analysis Zulli et al.34 Illumina Viral Surveillance Panel Illumina NovaSeq 6000 Chan-Zuckerberg Infectious Disease mNGS Illumina; minimap2; SPADES; bowtie2; NCBI NT/ NR databases ∼40 million reads per sample; 3%–90% percentage coverage, 5- to 20-fold read depth 33 respiratory panel viruses detected, enabling seasonal monitoring of outbreak timing and severity (including viruses with limited clinical surveillance) practical constraints necessitated sample pooling; varying coverage breadth and depth among targets (influenced by viral concentration/ outbreak activity); non-customized bioinformatics workflow limits analysis performance; matrix complexity affects sequencing performance (Continued on next page) 6 iS c ie n c e 2 9 , 1 1 7 5 9 6 , O c to b e r 1 6 , 2 0 2 6 iS cience R e v ie w ll O P E N A C C E S S iS c ie n c e 2 9 , 1 1 7 5 9 6 , O c to b e r 1 6 , 2 0 2 6 7 iS cience R e v ie w adapter ligation to the DNA template (Figure 2). ..
Activity Assay:Article Title: Sequencing the wastewater virome: Current strategies and bioinformatic pitfalls.
Article Snippet: .. Continued Study Target(s) Platform Pipeline/database Read count and depth/coverage (where reported) Key outcome Main limitations/ comments Probe-based targeted sequencing Karatas et al.42 TWIST Comprehensive Viral Panel AVITI EsViritu; Virus Pathogen Database > 3 million total reads mapped to viruses (n = 6; visual estimates from plots); reads were mapped with 90% percentage coverage criteria 2294 viral genomes or segments recovered, spanning 51 animal- and 135 human-associated viral species detection is biased toward known or closely related viruses (probe efficiency limited to sequences with > 80% nt similarity); limited assessment of viral population complexity (analysis focused on consensus genomes) Tisza et al.20 TWIST Comprehensive Viral Panel Illumina NovaSeq 6000 EsViritu; Virus Pathogen Database median 28.5 genomes/segments covered over > 90% of their length, additional 41 covered over > 50% of their length (percentage coverage; n = 363) broad community viral diversity: 465 virus strains detected with reads correlating with clinical data for some insufficient genome coverage for reliable allele/variant frequency analysis; low viral shedding limits coverage; Improved enrichment and increased depth are required to enhance coverage and variant analysis Zulli et al.34 Illumina Viral Surveillance Panel Illumina NovaSeq 6000 Chan-Zuckerberg Infectious Disease mNGS Illumina; minimap2; SPADES; bowtie2; NCBI NT/ NR databases ∼40 million reads per sample; 3%–90% percentage coverage, 5- to 20-fold read depth 33 respiratory panel viruses detected, enabling seasonal monitoring of outbreak timing and severity (including viruses with limited clinical surveillance) practical constraints necessitated sample pooling; varying coverage breadth and depth among targets (influenced by viral concentration/ outbreak activity); non-customized bioinformatics workflow limits analysis performance; matrix complexity affects sequencing performance (Continued on next page) 6 iS c ie n c e 2 9 , 1 1 7 5 9 6 , O c to b e r 1 6 , 2 0 2 6 iS cience R e v ie w ll O P E N A C C E S S iS c ie n c e 2 9 , 1 1 7 5 9 6 , O c to b e r 1 6 , 2 0 2 6 7 iS cience R e v ie w adapter ligation to the DNA template (Figure 2). ..
Adapter Ligation:Article Title: Sequencing the wastewater virome: Current strategies and bioinformatic pitfalls.
Article Snippet: .. Continued Study Target(s) Platform Pipeline/database Read count and depth/coverage (where reported) Key outcome Main limitations/ comments Probe-based targeted sequencing Karatas et al.42 TWIST Comprehensive Viral Panel AVITI EsViritu; Virus Pathogen Database > 3 million total reads mapped to viruses (n = 6; visual estimates from plots); reads were mapped with 90% percentage coverage criteria 2294 viral genomes or segments recovered, spanning 51 animal- and 135 human-associated viral species detection is biased toward known or closely related viruses (probe efficiency limited to sequences with > 80% nt similarity); limited assessment of viral population complexity (analysis focused on consensus genomes) Tisza et al.20 TWIST Comprehensive Viral Panel Illumina NovaSeq 6000 EsViritu; Virus Pathogen Database median 28.5 genomes/segments covered over > 90% of their length, additional 41 covered over > 50% of their length (percentage coverage; n = 363) broad community viral diversity: 465 virus strains detected with reads correlating with clinical data for some insufficient genome coverage for reliable allele/variant frequency analysis; low viral shedding limits coverage; Improved enrichment and increased depth are required to enhance coverage and variant analysis Zulli et al.34 Illumina Viral Surveillance Panel Illumina NovaSeq 6000 Chan-Zuckerberg Infectious Disease mNGS Illumina; minimap2; SPADES; bowtie2; NCBI NT/ NR databases ∼40 million reads per sample; 3%–90% percentage coverage, 5- to 20-fold read depth 33 respiratory panel viruses detected, enabling seasonal monitoring of outbreak timing and severity (including viruses with limited clinical surveillance) practical constraints necessitated sample pooling; varying coverage breadth and depth among targets (influenced by viral concentration/ outbreak activity); non-customized bioinformatics workflow limits analysis performance; matrix complexity affects sequencing performance (Continued on next page) 6 iS c ie n c e 2 9 , 1 1 7 5 9 6 , O c to b e r 1 6 , 2 0 2 6 iS cience R e v ie w ll O P E N A C C E S S iS c ie n c e 2 9 , 1 1 7 5 9 6 , O c to b e r 1 6 , 2 0 2 6 7 iS cience R e v ie w adapter ligation to the DNA template (Figure 2). ..
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