Journal: Genomics, Proteomics & Bioinformatics

Article Title: GREPore-seq: A Robust Workflow to Detect Changes After Gene Editing Through Long-range PCR and Nanopore Sequencing

doi: 10.1016/j.gpb.2022.06.002

Figure Lengend Snippet: A schematic overview of GREPore-seq workflow A . Step 1, laboratory process of cell culture, nucleofection, and gDNA extraction. B . Step 2, amplicon library preparation and nanopore sequencing. C . Step 3, GREPore-seq bioinformatic analysis. gDNA, genomic DNA; HSPC, hematopoietic stem and progenitor cell; RNP, ribonucleoprotein; sgRNA, single guide RNA; GREPore-seq, grepping reads of nanopore amplicon sequencing; dsODN, double-stranded oligodeoxynucleotide; HDR, homology-directed repair; BB, backbone.

Article Snippet: To evaluate the performance of KAPA HiFi DNA Polymerase (Catalog No. KK2602, Kapa Biosystems, Cape Town, South Africa), NileHiFi Long Amplicon PCR Kit (Catalog No. PC002, GeneCopoeia, Rockville, MD), and PrimeSTAR GXL DNA polymerase, each of the three long-range PCR enzymes was used to amplify the same wild-type gDNA sample.

Techniques: Cell Culture, Extraction, Amplification, Nanopore Sequencing, Sequencing

Journal: Genomics, Proteomics & Bioinformatics

Article Title: GREPore-seq: A Robust Workflow to Detect Changes After Gene Editing Through Long-range PCR and Nanopore Sequencing

doi: 10.1016/j.gpb.2022.06.002

Figure Lengend Snippet: Successful long-range PCR, trimming, and retrieval of full-length amplicon reads A . Comparison of three DNA polymerase kits. The quality and quantity of PCR products were assessed by electrophoresis on agarose gels. Specific primers (without barcode) for seven sites were used to amplify WT targets, each with two technical replicates. Red cross indicates failed amplification; red frames indicate expected products ( AAVS1 , 3928 bp; B2M , 5666 bp; BCL11A-1 , 8159 bp; BCL11A-2 , 8443 bp; EEF2 , 5287 bp; TRAC , 6485 bp; TRBC , 5093 bp). B . PCR success rates of PrimeSTAR, KAPA HiFi, and NileHiFi among 135 reactions. C . Removal of adaptors with Porechop. We used the command “seqkit locate -p” and the barcode “AACGGACT” (for BCL11A-3 primer-F) to detect the start location after Guppy or Porechop trimming. The two peaks in blue indicate nanopore sequencing adaptors. D . Distribution of nanopore raw read lengths before Porechop trimming. E . Distribution of nanopore read lengths after Porechop trimming. The percentages of read numbers were normalized to raw reads before Porechop processing. F . Strategy for retrieving full-length amplicon reads. A schematic of the Grepseq-left and Grepseq-right generations is shown. The retrieved BCL11A-3 amplicon reads were visualized with IGV after sampling 200 reads using the command “seqkit sample 200”. G . Distribution of nanopore read lengths after extracting the BCL11A-3 PCR product (3863 bp) by GERPore-seq. The read numbers were displayed as percentage of raw reads before trimming. WT, wild-type.

Article Snippet: To evaluate the performance of KAPA HiFi DNA Polymerase (Catalog No. KK2602, Kapa Biosystems, Cape Town, South Africa), NileHiFi Long Amplicon PCR Kit (Catalog No. PC002, GeneCopoeia, Rockville, MD), and PrimeSTAR GXL DNA polymerase, each of the three long-range PCR enzymes was used to amplify the same wild-type gDNA sample.

Techniques: Long Range PCR, Amplification, Comparison, Electrophoresis, Nanopore Sequencing, Sampling

Journal: Genomics, Proteomics & Bioinformatics

Article Title: GREPore-seq: A Robust Workflow to Detect Changes After Gene Editing Through Long-range PCR and Nanopore Sequencing

doi: 10.1016/j.gpb.2022.06.002

Figure Lengend Snippet: GREPore-seq effectively retrieves amplicon-specific nanopore reads with barcodes A . A schematic overview of the BC-primer-seqs with 10-nt or 12-nt barcodes. BC-primer-seqs are stretches of short overlapping sequences for data retrieval. B . and C. Higher data retrieval rate (B) and lower FDR (C) of GREPore-seq compared with those of Barcode_splitter.Data are represented as mean ± SEM ( n = 5 independent experiments). Paired two-sided Student’s t -tests were conducted. “ns” means no significance ( P > 0.05). FDR, false discovery rate.

Article Snippet: To evaluate the performance of KAPA HiFi DNA Polymerase (Catalog No. KK2602, Kapa Biosystems, Cape Town, South Africa), NileHiFi Long Amplicon PCR Kit (Catalog No. PC002, GeneCopoeia, Rockville, MD), and PrimeSTAR GXL DNA polymerase, each of the three long-range PCR enzymes was used to amplify the same wild-type gDNA sample.

Techniques: Amplification

Journal: Genomics, Proteomics & Bioinformatics

Article Title: GREPore-seq: A Robust Workflow to Detect Changes After Gene Editing Through Long-range PCR and Nanopore Sequencing

doi: 10.1016/j.gpb.2022.06.002

Figure Lengend Snippet: Strategy for retrieving full-length nanopore amplicon reads A . A schematic overview of Grepseqs and extraction for three distinct target reads. Bases and “–” marked in red indicate the mismatches aligned between the reference and a nanopore read. B . Pre-processing with Barcode_splitter decreases the data retrieval rate. Data were normalized to processing with GREPore-seq alone. The sequence of 4 nt or 5 nt at the beginning was used in Barcode_splitter. 20:90 or 20:150 represents the range of Grepseqs used for amplicon-specific read extraction. The data were statistically analyzed by two-way ANOVA (adjusted P values were indicated). C . Low misassignment of amplicon reads by GREPore-seq. Numbers in red indicate erroneously assigned reads.

Article Snippet: To evaluate the performance of KAPA HiFi DNA Polymerase (Catalog No. KK2602, Kapa Biosystems, Cape Town, South Africa), NileHiFi Long Amplicon PCR Kit (Catalog No. PC002, GeneCopoeia, Rockville, MD), and PrimeSTAR GXL DNA polymerase, each of the three long-range PCR enzymes was used to amplify the same wild-type gDNA sample.

Techniques: Amplification, Extraction, Sequencing

Journal: Genomics, Proteomics & Bioinformatics

Article Title: GREPore-seq: A Robust Workflow to Detect Changes After Gene Editing Through Long-range PCR and Nanopore Sequencing

doi: 10.1016/j.gpb.2022.06.002

Figure Lengend Snippet: GREPore-seq correctly identifies short dsODN insertions A. Top: A schematic overview of 29-bp dsODN insertions at CRISPR/Cas9-mediated DSBs and generation of DSgrep-seqs for retrieving reads with the insertion. Bottom: Visualization of WT- or dsODN-inserted-amplicon reads and showcase of insertion details. Bases marked in red and blue indicate forward and reverse dsODN insertions, respectively; dark-red bases indicate recurring alignment; bases and “–” marked in black indicate mismatches between 29-bp dsODN and the nanopore read, likely due to basecall errors. B. and C. Effects of DSgrep-seq lengths on retrival rate (B) and FDR (C) of reads with dsODN insertions. Editing without dsODN serves as a control to determine the FDR. Data are represented as mean ± SEM ( n = 24−26 independent experiments). D. and E. Effects of step values on retrieval rate (D) and FDR (E) of reads with dsODN insertions. F. Comparison of dsODN retrieval rates analyzed by three methods. G. Correlation of dsODN retrieval rates analyzed by CRISPResso2, GrepNGS, and GREPore-seq. The data in (B–F) were statistically analyzed by two-way ANOVA (adjusted P values were indicated). “ns” means no significance ( P > 0.05). DNA double-strand break; WT, wild-type; GrepNGS, NGS data analyzed by both CRISPResso2 and GREPore-seq.

Article Snippet: To evaluate the performance of KAPA HiFi DNA Polymerase (Catalog No. KK2602, Kapa Biosystems, Cape Town, South Africa), NileHiFi Long Amplicon PCR Kit (Catalog No. PC002, GeneCopoeia, Rockville, MD), and PrimeSTAR GXL DNA polymerase, each of the three long-range PCR enzymes was used to amplify the same wild-type gDNA sample.

Techniques: CRISPR, Amplification, Control, Comparison

Journal: The ISME Journal

Article Title: A 'universal' type II chaperonin PCR detection system for the investigation of Archaea in complex microbial communities

doi: 10.1038/ismej.2011.96

Figure Lengend Snippet: PCR primers used in this study

Article Snippet: Thermosome PCR amplicons (JH0175/JH0178 only) from the dry food diet were also subjected to pyrosequencing.

Techniques: Sequencing

Journal: The ISME Journal

Article Title: A 'universal' type II chaperonin PCR detection system for the investigation of Archaea in complex microbial communities

doi: 10.1038/ismej.2011.96

Figure Lengend Snippet: Nucleotide frequencies for the thermosome (group II chaperonin) primer annealing sites. The frequency of each nucleotide in each position in the 166 sequence alignment is indicated. Where more than two different nucleotides were common, inosine (I) was used in JH0175/JH0178 to reduce degeneracy. The graphs are shown such that the x-axis depicts the primer sequences in their 5′–3′ orientation. The ‘high GC' JH0268/JH0269 primers are depicted directly below their counterparts to highlight how and where degeneracy was removed.

Article Snippet: Thermosome PCR amplicons (JH0175/JH0178 only) from the dry food diet were also subjected to pyrosequencing.

Techniques: Sequencing

Journal: The ISME Journal

Article Title: A 'universal' type II chaperonin PCR detection system for the investigation of Archaea in complex microbial communities

doi: 10.1038/ismej.2011.96

Figure Lengend Snippet: Thermosome PCR products from (a) the JH0175/JH0178 primer set, (b) the JH0268/JH0269 primer set and (c) the 7:1 cocktail of JH0175/JH0178:JH0268/JH0269 using 1.0 ng per reaction of genomic DNA from archaeal isolates as templates. The GC content of the species (or genus range, if no complete genome sequence is available for that species) is given in parentheses after the species name. Lanes are (NTC) PCR no template control; (Neg) Escherichia coli DH5α (1) Mc. voltae (28% GC); (2) Mc. vannielii (31% GC); (3) Mc. maripaludis (33% GC); (4) Mt. igneus (38% GC); (5) Ms. hungarei (45% GC); (6) S. solfataricus (36% GC); (7) Sulfolobus sp. (33–36% GC); (8) Tp. acidophilum (46% GC); (9) Tc. gorgonarius (40–54% GC); (10) Tc. pacificus (40–54% GC); (11) Tc. zilligii (40–54% GC); (12) Hb. salinarum (formerly Hb. halobium) (66% GC); (13) Hb. salinarum (formerly Hb. cutirubrum) (66% GC); (14) Hb. salinarum (formerly Hb. salinarium) (66% GC); (15) Hf. volcanii WR341 (66% GC) and (16) Hf. volcanii WR536 (66% GC).

Article Snippet: Thermosome PCR amplicons (JH0175/JH0178 only) from the dry food diet were also subjected to pyrosequencing.

Techniques: Sequencing

Journal: The ISME Journal

Article Title: A 'universal' type II chaperonin PCR detection system for the investigation of Archaea in complex microbial communities

doi: 10.1038/ismej.2011.96

Figure Lengend Snippet: Phylogenetic trees of DNA sequences identified as Mb. smithii from rumen clone libraries based on the (a) 16S rRNA gene, (b) mcrA gene or (c) thermosome gene (‘universal' JH0175/JH0178 amplification only). Trees are neighbour-joined consensus trees based on 100 replicates. Nodes with bootstrap values greater than 50 (*) or 90 (**) are indicated. Mb. smithii reference sequences were taken from strain DSM 2374 and Mc. maripaludis C5 sequences were used as outliers. Numbers in parentheses indicate how many times a sequence was detected in the library when present more than once.

Article Snippet: Thermosome PCR amplicons (JH0175/JH0178 only) from the dry food diet were also subjected to pyrosequencing.

Techniques: Amplification, Sequencing

Journal: PLoS ONE

Article Title: Nanopore sequencing of SARS-CoV-2: Comparison of short and long PCR-tiling amplicon protocols

doi: 10.1371/journal.pone.0259277

Figure Lengend Snippet: In both runs, an initial portion of the run containing on average 40-Mbp of sequencing data per barcode was used. Coverage values higher than 1000 were clipped at this value and are shown in blue. Coverage below 20 (default Artic cutoff) is shown in red. Medians of 10-bp windows are shown for smoothing. The very starts and ends of the genome are not covered by amplicons and are thus displayed in red. Shaded area in the left column corresponds to amplicon 13. Some barcodes have a visible dip in the coverage at the left end of this amplicon; this difference in coverage is caused by reads originating from sub-genomic RNAs corresponding to the gene S. Similar plots for additional runs are shown in .

Article Snippet: The combination of PCR-tiling of overlapping amplicon pools with nanopore sequencing on the MinION platform from Oxford Nanopore Technologies is one of the most powerful and versatile means for acquisition of viral sequences.

Techniques: Sequencing, Amplification