Journal: bioRxiv

Article Title: Artificial intelligence-driven morphology-based enrichment of malignant cells from body fluid

doi: 10.1101/2023.01.24.525423

Figure Lengend Snippet: Table of sample cases showing pre- and post-sort somatic variant allele frequency (VAF), as detected by targeted mutation sequencing (56G oncogene panel). Primary mutation profiles from tissue and/or IHC for corresponding sample cases are shown, with orange highlighted text indicating matching detected mutation in post-sort specimens determined by Ampliseq Cancer Hotspot panel V2.

Article Snippet: Target amplification for mutation detection was performed using 20 ng of WGA product with Integrated DNA Technologies xGenTM Amplicon Core kit and xGenTM 56G Onco Amplicon Panel v2 (Integrated DNA Technologies), which offers comprehensive and hotspot coverage of 56 clinically-relevant oncology-related genes (ABL1, AKT1, ALK, APC, ATM, BRAF, CDH1, CDKN2A, CSF1R, CTNNB1, DDR2, DNMT3A, EGFR, ERBB2, ERBB4, EZH2, FBXW7, FGFR1, FGFR2, FGFR3, FLT3, FOXL2, GNA11, GNAQ, GNAS, HNF1A, HRAS, IDH1, IDH2, JAK2, JAK3, KDR, KIT, KRAS, MAP2K1, MET, MLH1, MPL, MSH6, NOTCH1, NPM1, NRAS, PDGFRA, PIK3CA, PTEN, PTPN11, RB1, RET, SMAD4, SMARCB1, SMO, SRC, STK11, TP53, TSC1, VHL).

Techniques: Variant Assay, Mutagenesis, Sequencing

Journal: bioRxiv

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

doi: 10.1101/2021.12.13.472514

Figure Lengend Snippet: 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.

Article Snippet: To evaluate the performance of KAPA HiFi DNA Polymerase (Kapa Biosystems), NileHiFi Long Amplicon PCR Kit (GeneCopoeia), and PrimeSTAR, each of the three long-range PCR enzymes was used to amplify the same wild-type genomic DNA sample.

Techniques: Cell Culture, Extraction, Amplification, Nanopore Sequencing

Journal: bioRxiv

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

doi: 10.1101/2021.12.13.472514

Figure Lengend Snippet: 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 wild-type targets, each with two technical replicates. Red cross, failed amplification; red frames, 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 , The 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 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. Visualization of retrieved BCL11A-3 amplicon reads 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 normalized to raw reads before trimming.

Article Snippet: To evaluate the performance of KAPA HiFi DNA Polymerase (Kapa Biosystems), NileHiFi Long Amplicon PCR Kit (GeneCopoeia), and PrimeSTAR, each of the three long-range PCR enzymes was used to amplify the same wild-type genomic DNA sample.

Techniques: Comparison, Electrophoresis, Amplification, Nanopore Sequencing, Sampling

Journal: bioRxiv

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

doi: 10.1101/2021.12.13.472514

Figure Lengend Snippet: A , A schematic overview of Grepseqs and extraction for three distinct target reads. Bases and “-” that are marked in red indicate the mismatches aligned between the reference and a nanopore read. B , Preprocessing with Barcode_splitter decreases the data recovery rate. Data were normalized to GREPore-seq alone. The sequence of 4 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. C , Low misassignment of amplicon reads by GREPore-seq. The data in B were statistically analyzed by two-way ANOVA. Adjusted P values were indicated.

Article Snippet: To evaluate the performance of KAPA HiFi DNA Polymerase (Kapa Biosystems), NileHiFi Long Amplicon PCR Kit (GeneCopoeia), and PrimeSTAR, each of the three long-range PCR enzymes was used to amplify the same wild-type genomic DNA sample.

Techniques: Extraction, Sequencing, Amplification

Journal: bioRxiv

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

doi: 10.1101/2021.12.13.472514

Figure Lengend Snippet: A , Top: A schematic overview of dsODN 29 bp insertions at DSBs with Cas9-gRNA RNP gene editing and generation of DSgrep-seqs for retrieving reads with the insert. Bottom: Visualization of WT- or dsODN-amplicon reads and amplification of insertion details of dsODN data. Bases marked in red and blue indicate forward and reverse dsODN insertions; dark-red bases indicate recurring alignment; bases and “-” marked in dark indicate mismatches aligned between 29 nt dsODN and a nanopore read. BC , Effects of DSgrep-seq lengths and step values on retrieval rate (B) and FDR (C) of reads with dsODN. Editing without dsODN serves as a control to determine the false retrieval rate (C). Error bars represent the mean ± SEM of n = 24 - 26 independent experiments. DE , Step values ranging from 1 to 5 moderately affect the data recovery rate (D) and FDR (E). F , dsODN insertion rates were analyzed by three methods. G - I , dsODN retrieval rates analyzed by CRISPREsso2, GrepNGS, and GREPore-seq from data in B and D are highly correlated. The data in B-F were statistically analyzed by two-way ANOVA. Adjusted P values were indicated. “ns” means no significance ( p > 0.05).

Article Snippet: To evaluate the performance of KAPA HiFi DNA Polymerase (Kapa Biosystems), NileHiFi Long Amplicon PCR Kit (GeneCopoeia), and PrimeSTAR, each of the three long-range PCR enzymes was used to amplify the same wild-type genomic DNA sample.

Techniques: Amplification, Control

Journal: Genome Biology

Article Title: Biased estimates of clonal evolution and subclonal heterogeneity can arise from PCR duplicates in deep sequencing experiments

doi: 10.1186/s13059-014-0420-4

Figure Lengend Snippet: Adaptation of Illumina TruSeq Custom Amplicon Kit to allow for single molecule tagging. (A) Schematic of method showing amplification of target DNA using custom probes and flanking primers. The P5-SMT primer is the same as the standard P5 index primer, but contains a degenerate 12 N-mer sequence in place of the index. The incorporation of an Ampure Bead size selection step after two rounds of PCR removes unused P5-SMT, and the P5 primer is added to facilitate downstream amplification. Figure schematic is adapted from Illumina promotional material. (B) Stacked barplot showing number of paired-end reads, split into unique reads (dark grey) and SMT-identified duplicate reads (light-grey) in 24 samples (18 germline, 6 tumor). (C) For each of 18 germline samples, we show the number of SMTs by duplicate cluster size (the number of times that an SMT was observed at a given target within a sample). Higher overall duplicate rates within a sample were associated with larger duplicate clusters. Except for the sample with the highest duplicate rate, duplicate cluster sizes were generally less than 10. The length of the SMT (8- versus 12-mer) did not affect the distribution. SMT, single molecule tag.

Article Snippet: This indicates that our method effectively incorporates diverse SMT oligomers into the library preparation process and suggests that the Illumina TruSeq Custom Amplicon Kit generates a moderate number of PCR duplicate reads that are evenly distributed across targeted sequences.

Techniques: Amplification, Sequencing, Selection

Journal: BMC Genomics

Article Title: A rapid, cost-effective tailed amplicon method for sequencing SARS-CoV-2

doi: 10.1186/s12864-020-07283-6

Figure Lengend Snippet: Methods for SARS-CoV-2 genome sequencing compared in this study. a In Illumina’s Nextera DNA Flex Enrichment protocol cDNA is tagmented and made into barcoded sequencing libraries, which are then enriched using sequence capture with a respiratory virus panel containing probes against SARS-CoV-2. b In the ARTIC protocol, first strand cDNA is enriched by amplifying with two pools of primers to generate amplicons tiling the SARS-CoV-2 genome. These amplicons are then subjected to either Illumina or Oxford Nanopore library preparation, using methods that either directly add adapters to the ends of the amplicons or to fragment them to enable sequencing on a wider variety of Illumina instruments. c The tailed amplicon approach, developed here, enriches first strand cDNA using ARTIC v3 primers containing adapter tails. This allows functional sequencing libraries to be created through a second indexing PCR reaction that adds sample-specific barcodes and flow cell adapters

Article Snippet: Amplicon libraries (ARTIC v3, Tailed v1, Tailed v2) were diluted to 8 pM in Illumina’s HT1 buffer, spiked with 5% PhiX, and sequenced using a MiSeq 600 cycle v3 kit (Illumina, San Diego, CA).

Techniques: Sequencing, Virus, Amplification, Functional Assay

Journal: BMC Genomics

Article Title: A rapid, cost-effective tailed amplicon method for sequencing SARS-CoV-2

doi: 10.1186/s12864-020-07283-6

Figure Lengend Snippet: Comparison of sequence capture, ARTIC v3 amplicon, and tailed amplicon workflows on SARS-CoV-2 isolate. a Percentage of the BEI WA1 isolate genome coverage at 10x at different subsampled read depths when sequenced with the indicated approach. b Percent of the BEI WA1 isolate genome coverage at 100x at different subsampled read depths when sequenced with the indicated approach. c Observed read depth for each of the expected amplicons for the BEI WA1 isolate amplified with the ARTIC v3 protocol at a subsampled read depth of 100,000 raw reads. d Observed read depth for each of the expected amplicons for the BEI WA1 isolate amplified with the tailed amplicon v1 (2 pool amplification) protocol at a subsampled read depth of 100,000 raw reads. e Observed read depth for each of the expected amplicons for the BEI WA1 isolate amplified with the tailed amplicon v2 protocol (4 pool amplification) at a subsampled read depth of 100,000 raw reads. f Variants detected for the BEI WA1 isolate at a read depth of up to 1,000,000 raw reads (or the maximum read depth for the sample if below 1,000,000 reads). The positions of all variants detected in this study are shown and bases where the sample matches the Wuhan-Hu-1 reference are shown in grey

Article Snippet: Amplicon libraries (ARTIC v3, Tailed v1, Tailed v2) were diluted to 8 pM in Illumina’s HT1 buffer, spiked with 5% PhiX, and sequenced using a MiSeq 600 cycle v3 kit (Illumina, San Diego, CA).

Techniques: Comparison, Sequencing, Amplification

Journal: BMC Genomics

Article Title: A rapid, cost-effective tailed amplicon method for sequencing SARS-CoV-2

doi: 10.1186/s12864-020-07283-6

Figure Lengend Snippet: Comparison of sequence capture, ARTIC v3 amplicon, and tailed amplicon workflows on clinical specimens spanning a range of viral loads. a Samples with N1 and N2 Ct values ranging from approximately 20–35 chosen for testing of SARS-CoV-2 sequencing workflows. Samples are colored as in panels c - f . b Evenness of representation of amplicons for different workflows as a function of sample N1 Ct value. Percentage of genome coverage at 100x at different subsampled read depths for each sample when sequenced using the following approaches: c Illumina Nextera DNA Enrichment; d ARTIC v3 with TruSeq library preparation. e Tailed amplicon v1 (2 pool amplification); f Tailed amplicon v2 (4 pool amplification)

Article Snippet: Amplicon libraries (ARTIC v3, Tailed v1, Tailed v2) were diluted to 8 pM in Illumina’s HT1 buffer, spiked with 5% PhiX, and sequenced using a MiSeq 600 cycle v3 kit (Illumina, San Diego, CA).

Techniques: Comparison, Sequencing, Amplification