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