Library preparation and target enrichment standard hybridization workflow followed the guidelines of Twist Next Generation Sequencing (NGS) protocols Twist Total Nucleic Acids Library Preparation EF Kit 2.0 for Viral Pathogen Detection and Characterization along with their Twist Target Enrichment Protocol. Three previously extracted RNA samples were diluted along with synthetic controls. cDNA synthesis and purification followed. Next, DNA fragmentation, telomere repair, and dA-Tailing were performed. Universal Twist adapters were then ligated to the cDNA and purified. Finally, PCR amplification was conducted to index the samples and finish the library preparation portion of the study. A single pooled library was first prepared from the indexed library-prepped sample pools. This was followed by hybridization of the targets in solution, which was ~16 h in total to complete. Next, the binding of hybridized targets to desired streptavidin beads occurred. Libraries were then enriched via PCR amplification and purification utilizing 23 cycles as recommended by Twist Technical Support. Sample libraries were ready for sequencing on the Illumina NGS platform according to manufacturer protocols after PCR amplification and purification. Sequencing data was processed on the One Codex bioinformatics online platform and according to methods described below.
Ngs platform
Manufactured by Illumina
Sourced in United States
The NGS platform is a high-throughput DNA sequencing system designed for scientific research. It utilizes next-generation sequencing (NGS) technology to enable rapid and accurate analysis of genetic material. The core function of the NGS platform is to perform parallel sequencing of DNA or RNA samples, providing comprehensive genomic data for a wide range of applications.
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Lab products found in correlation
Biosprint 96 dna plant kit, by Qiagen (3 mentions)
Truseq pe cluster kit, by Illumina (2 mentions)
Truseqtm nano dna lt sample prep kit set a, by Illumina (2 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (2 mentions)
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (1 mentions)
Qubit fluorometer 2, by Thermo Fisher Scientific (1 mentions)
Agilent dna 1000 chip, by Agilent Technologies (1 mentions)
2100 bioanalyzer, by Thermo Fisher Scientific (1 mentions)
Nextera xt index kit, by Illumina (1 mentions)
Qiaamp dna mini kit, by Qiagen (1 mentions)
Rnasimple total rna kit, by Tiangen Biotech (1 mentions)
Next ultra 2 kit, by Illumina (1 mentions)
E220 ultrasonicator, by Covaris (1 mentions)
Dead cell removal kit, by Miltenyi Biotec (1 mentions)
Casava v1, by Illumina (1 mentions)
Pe sample preparation kit, by Illumina (1 mentions)
Truseq nano dna lt library prep kit, by Illumina (1 mentions)
Ampure xp, by Beckman Coulter (1 mentions)
Qiaseq fx single cell rna library kit, by Qiagen (1 mentions)
Agencourt ampure xp magnetic beads, by Beckman Coulter (1 mentions)
32 protocols using ngs platform
1
Twist NGS Library Prep for Viral Detection
2
NGS-based Profiling of TGF-β Pathway Mutations
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The pathological diagnosis of the specimens was confirmed by hematoxylin and eosin (HE) staining. To be considered as a qualified sample, the specimen needed to be ≥1 mm and the percentage of tumor cells needed to be over 20%. Subsequently, 50–200 ng of DNA extracted from the samples were broken into ~200 bp fragments and then subjected to NGS platform Illumina (San Diego, CA, USA) Nextseq 500 to >500× coverage (24 (link)).
Tumor mutation burden (TMB) was defined as somatic mutation counts in coding region per megabase of genome examined. Genes including TGFBR2, ACVR1B, ACVR2A, INHBA, SMAD2, SMAD3, SMAD4, IGF2, RUNX1, STAT3, TERT, and VEGFA have been reported to be the core members in the TGF-β pathway network and to act as cancer-related genes (21 (link)); they were thus selected to be included for analysis. A TGF-β pathway mutation was defined as the presence of at least one pathway gene with identified mutational types (missense mutation or truncating mutation), including nonsense mutation, nonstop mutation, frameshift insertion, frameshift deletion, and splice site mutation.
Tumor mutation burden (TMB) was defined as somatic mutation counts in coding region per megabase of genome examined. Genes including TGFBR2, ACVR1B, ACVR2A, INHBA, SMAD2, SMAD3, SMAD4, IGF2, RUNX1, STAT3, TERT, and VEGFA have been reported to be the core members in the TGF-β pathway network and to act as cancer-related genes (21 (link)); they were thus selected to be included for analysis. A TGF-β pathway mutation was defined as the presence of at least one pathway gene with identified mutational types (missense mutation or truncating mutation), including nonsense mutation, nonstop mutation, frameshift insertion, frameshift deletion, and splice site mutation.
3
Fungal Metagenomic Sequencing with Nextera XT
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The amplicon libraries were prepared using Nextera XT Index Kit (Illumina Inc., San Diego, California, USA) as per the ITS Metagenomic Sequencing Library preparation protocol (Part # 15044223 Rev. B). The amplicons with the Illumina adaptors were amplified using i5 and i7 primers that add multiplexing index sequences as well as common adapters required for cluster generation (P5 and P7). The amplicon libraries were purified by 1X AMpureXP beads, checked on Agilent DNA 1000 chip on Bioanalyzer 2100 and quantified by Qubit Fluorometer 2.0 using Qubit dsDNA HS Assay kit (Life Technologies, India). After obtaining the Qubit concentration for the library and the mean peak size from Bioanalyser profile, the library was spiked with 50% PhiX control v3 (FC-110-3001) as described in the Illumina procedure and loaded onto illumina NGS platform at an appropriate concentration (10–20 pM) for cluster generation and sequencing. The libraries were sequenced at Xcelris Genomics Pvt. Ltd. (Ahmedabad, India), using Illumina HiSeq 2 X 250 base pair chemistry. Sequencing of PCR negative reactions did not yield any fungal reads.
4
Illumina NGS Protein Interaction Protocol
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Data were collected from an Illumina NGS platform using standard library and sequencing chemistry methods for paired 150 bp read lengths per the manufacturers' recommended protocol. Fastq files of paired‐end sequences were aligned to a sequence database of the human protein interaction reference set and read counts for each fragment pair were normalized and scaled using methods as described previously.46 Read counts were statistically analyzed in the EdgeR package as previously described.46 Interacting fragment pairs were determined using cutoffs of Log2 fold change of the normalized read counts of greater than or equal to 1 and a False Discovery Rate of <10%.
5
Genome Sequencing of M. purpureus CSU-M183
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The whole genome of the M. purpureus CSU-M183 strain was sequenced using SMRT sequencing technology of PacBioRS II, and the sequencing quality was improved using Illumina NGS platform. The sequencing library was constructed using the TruSeqTM Nano DNA LT Sample Prep Kit–Set A (Illumina, USA) and amplified using the TruSeq PE Cluster Kit (Illumina, USA).
The quality of the assembled genome and annotated geneset were assessed first using the Benchmarking Universal Single-Copy Orthologs (version 3.1.0; BUSCO) with the fungi_odb9 dataset [33 (link)].
For raw data polymerase reads after PacBioRS II sequencing, subreads were obtained by removing the low-quality or unknown reads, adapters and duplications. The filtered reads were assembled de novo using the Hierarchical Genome Assembly Process (HGAP) algorithm version 2.0 [34 (link)].
For genome assembly, the default parameters of HGAP2 were used (Minimum Subread Length = 500, Minimum Polymerase Read Quality = 0.80, Minimum Polymerase Read Length = 100, Overlapper Error Rate = 0.06, Overlapper Min Length = 40) with input genome size as 30 Mb.
The quality of the assembled genome and annotated geneset were assessed first using the Benchmarking Universal Single-Copy Orthologs (version 3.1.0; BUSCO) with the fungi_odb9 dataset [33 (link)].
For raw data polymerase reads after PacBioRS II sequencing, subreads were obtained by removing the low-quality or unknown reads, adapters and duplications. The filtered reads were assembled de novo using the Hierarchical Genome Assembly Process (HGAP) algorithm version 2.0 [34 (link)].
For genome assembly, the default parameters of HGAP2 were used (Minimum Subread Length = 500, Minimum Polymerase Read Quality = 0.80, Minimum Polymerase Read Length = 100, Overlapper Error Rate = 0.06, Overlapper Min Length = 40) with input genome size as 30 Mb.
6
Assessing Off-Target Effects in iACBE Edits
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To analyze the off-target effects induced by the iACBE system, total genomic DNA was extracted from the laboratory DH5α strain and three edited clones (iACBE4 with galK gRNA1, iACBE4-NG with galK NG-gRNA1 containing native or esgRNA scaffold) using QIAamp DNA minikit (Qiagen, Germany). Whole-genome sequencing was performed at SEEDERS Inc. (SEEDERS, Daejeon, South Korea) with the Illumina NGS platform. Raw sequence reads were aligned to the reference E. coli genome (NCBI accession: GCA_022221385.1 ) or laboratory DH5α strain. The Illumina reads were preprocessed using Trimmomatic (v. 0.39) (54 (link)). SEEDERS in-house script (55 (link)) was utilized to map SNVs using BWA (0.7.17 r1188) and SAMtools (v 0.1.16) programs (56 (link), 57 ). The alignment accuracy to the reference genome was 99.84% for all four samples. The SNV for a nucleobase was counted as homozygous (read rate 90%) or heterozygous (40%≤ read rate ≤60%) based on the read rate. Mutation calls found in all the samples and parent DH5α were excluded and not considered off-targets.
7
Transcriptome Analysis of Aphid Lucorum
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Total RNA from whole bodies of A. lucorum was extracted using the RNA simple Total RNA Kit (Tiangen, China) according to the manufacturer’s instructions, and then sequenced with the Illumina NGS platform to generate high-throughput RNA sequencing (RNA-Seq) data. The resultant raw reads were processed by removing poor quality reads and trimming adaptors. In the absence of a reference genome of A. lucorum, the clean reads were imported into the Trinity assembler with the default parameters for de novo assembly (Grabherr et al., 2011 ). For the assembled transcripts, the TransDecoder program was used to identify candidate coding regions. Signal peptides were predicted using SignalP v3.0 (Bendtsen et al., 2004 (link)). The aphid effector sequences were retrieved from publications (Bos et al., 2010 (link); Atamian et al., 2013 (link)), and used as queries for Blast searches (E-value <1×10–5) against A. lucorum secreted proteins. The domain component in each protein sequence was predicted using the Pfam database (Finn et al., 2016 (link)). To predict GPx domain-containing proteins in each insect, the hidden Markov model profile of GPx (PF00255) was obtained from the Pfam database, and then used to perform a HMM search against insect proteins using the HMMER program (Finn et al., 2011 (link)).
8
In-Situ Hi-C of Xenograft Tumors
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18×106 tumor cells from P21_ADA patient were purified with Dead Cell Removal Kit (Miltenyi Biotec 130-090-101) according to the manufacturer’s instruction reaching a viability of 92%. Two aliquots of 4×106 purified tumor cells and FACS sorted tumor cells derived from spleen and bone marrow of xenotransplants were subjected to in-situ Hi-C with the Arima Hi-C kit (Arima, San Diego, CA, USA) following manufacturer instructions. Briefly, PBS-resuspended cells were crosslinked with 1% formaldehyde at room temperature for 10 minutes and reaction was stopped following user’s instruction. For each generated HiC library, two tubes with 1.2 to 1.4 ug of library were sonicated by Covaris E220 ultrasonicator at the following conditions: 10% Duty factor, 200 cycle, 140 peak, 63 seconds. Biotin-enriched fragments were size-selected with an average size of 400 bp and subjected to end-repaired with the NEB Next Ultra-II kit (E7645L) and tagged with different Illumina DNA adapters. Libraries were amplified with 7 cycles following KAPA HyperPrep PCR conditions (07962347001) and verified by q-PCR with KAPA-Q-PCR reagents (07960140001). Libraries were sequences by Illumina NGS platform with paired-end sequencing at 300 cycles to reach a sequencing depth of 40x to 50x.
9
Genomic DNA Isolation and Genotyping
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All blood samples will be transferred to the central laboratory in Isfahan Cardiovascular Research Institute. Using the salting-out approach, DNA was isolated from 500 µl of peripheral blood17 (link). Nanodrop and agarose gel were then used to assess the quality of the DNA. In the next step, primers with the following sequences were designed to genotype exons 21 and 26: Forward 5-TCTCATGAAGGTGAGTTTTC-3, and reverse 5-AGAGCATAGTAAGCAGTAGG-3; Forward 5-CCTTAATCTCACAGTAACTTGGCA-3, and reverse 5-AAGGGTGTGATTTGGTTGCT-3, respectively. The PCR materials included 1.6 μL DNA, 20 μL of mastermix (Ampliqon A/S co.), 0.7 l of each primer, and distiled water up to 40 μL with the following instructions: 95 °C for 5 min, 30 cycles of 95 °C for 45 s, 59 °C for 40 s, and 72 °C for 30 s, and 72 °C for 80 s as the final extension. Finally, the PCR products were sequenced bidirectionally by the Sanger technique utilizing Device (Illumina NGS platform) after confirming the quality and quantity on the gel electrophoresis.
10
Multiplex KIR Genotyping by NGS
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