Somatic mutations were identified by the Strelka2 variant caller with default parameters18 (link). Variants were subsequently annotated by wAnnovar19 (link). Tumor mutation burden was estimated based on the proportion of nonsynonymous single nucleotide variants and short indels per coding megabase. Mutational signature analysis was performed with mSigAct v2.0.0.901820 (link). All mutational signatures detected previously in whole-exome sequencing data were considered for initial mutational signature assignment using the SparseAssignActivity function, with a p.thresh at 0.5 to reduce stringency21 (link). Mutational signature assignment was done after adjusting the signatures for exome trinucleotide abundance. To validate variants by Sanger sequencing, PCR amplicons encompassing the mutation sites were sequenced using the ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction kit (Applied Biosystems, Foster City, CA, USA) on an ABI 3730 xl DNA Analyzer (Applied Biosystems). Functional impact of variants were predicted using published tools22 (link)–24 (link).
Abi prism bigdye terminator cycle sequencing ready reaction kit
Manufactured by Thermo Fisher Scientific
Sourced in United States, China, Germany
The ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kit is a DNA sequencing reagent kit. It provides the necessary components for dye-terminator cycle sequencing reactions.
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Lab products found in correlation
Abi 3730xl genetic analyzer, by Thermo Fisher Scientific (9 mentions)
Wizard sv gel and pcr clean up kit, by Promega (9 mentions)
Qiaquick pcr purification kit, by Qiagen (8 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (7 mentions)
Abi prism 310 genetic analyzer, by Thermo Fisher Scientific (7 mentions)
Qiaamp viral rna kit, by Qiagen (5 mentions)
Qiaquick gel extraction kit, by Qiagen (5 mentions)
Abi prism 3730 genetic analyzer, by Thermo Fisher Scientific (5 mentions)
Abi 3730xl dna analyzer, by Thermo Fisher Scientific (4 mentions)
Abi prism 3100 genetic analyzer, by Thermo Fisher Scientific (4 mentions)
Abi prism 377 dna sequencer, by Thermo Fisher Scientific (3 mentions)
Abi 3730xl sequencer, by Thermo Fisher Scientific (2 mentions)
3730 nucleotide sequencer, by Thermo Fisher Scientific (2 mentions)
Qiaex 2 gel extraction kit, by Qiagen (2 mentions)
Qiaamp dna blood mini kit, by Qiagen (2 mentions)
3100 avant genetic analyzer, by Thermo Fisher Scientific (2 mentions)
Abi3500 automated sequencer, by Thermo Fisher Scientific (2 mentions)
Abi 3100 genetic analyzer, by Thermo Fisher Scientific (2 mentions)
Sequencher version 4, by Gene Codes (2 mentions)
Nucleospin plant kit, by Macherey-Nagel (2 mentions)
133 protocols using abi prism bigdye terminator cycle sequencing ready reaction kit
1
Comprehensive Genomic Profiling Pipeline
2
DENV Genome Sequencing and Analysis
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The nucleotide sequence of the genome region encoding the envelope was determined by Sanger sequencing using an ABI sequencer. The sequencing reactions were prepared using an ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction kit, version 3.1 (Applied Biosystems, Vilnius, Lithuania). Briefly, 2 μL of DNA template was mixed with 2 μL of reaction mix, 1 μL of 5× sequencing buffer, 1.6 μL of specific primer (Table S1 ), and 3.4 μL of nuclease-free water. The amplification cycle conditions were programmed according to the manufacturer’s protocol.
The whole genome sequences of DENV isolates obtained in the present study were determined by next-generation sequencing (NGS) on a Miseq platform (Illumina, San Diego, CA, USA). Preparation of the NGS library using NexteraXT (Illumina, San Diego, CA, USA) was described previously [8 (link)]. The FASTQ results were examined using CLC Genomics Workbench software, version 21 (CLC Bio, QIAGEN, Valencia, CA, USA). Whole-genome assembly was conducted using Map read reference command (DENV-1 Mochizuki AB074760.1 and DENV-2 16681 NC_001474.2 were used as reference strains).
All newly obtained sequences of the envelope and the whole genome were deposited in GenBank with accession numbers OQ832560-OQ832594 and OQ832609-OQ832627, respectively.
The whole genome sequences of DENV isolates obtained in the present study were determined by next-generation sequencing (NGS) on a Miseq platform (Illumina, San Diego, CA, USA). Preparation of the NGS library using NexteraXT (Illumina, San Diego, CA, USA) was described previously [8 (link)]. The FASTQ results were examined using CLC Genomics Workbench software, version 21 (CLC Bio, QIAGEN, Valencia, CA, USA). Whole-genome assembly was conducted using Map read reference command (DENV-1 Mochizuki AB074760.1 and DENV-2 16681 NC_001474.2 were used as reference strains).
All newly obtained sequences of the envelope and the whole genome were deposited in GenBank with accession numbers OQ832560-OQ832594 and OQ832609-OQ832627, respectively.
3
Genetic analysis of SLC29A3 exons
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Peripheral blood samples from the family members and 100 population-matched unrelated healthy control individuals were collected following informed consent and Institute Ethics Committee approval. Genomic DNA was isolated from peripheral blood leucocytes according to standard techniques.
All 6 exons of SLC29A3 were amplified by polymerase chain reaction (PCR) from the genomic DNA using Premix LA Taq (Takara Biotechnology Co., Dalian, China) and previously reported primers.[2 (link)] The amplified PCR products were directly sequenced on an ABI Prism 3730×l Automated Sequencer, using the ABI Prism Big Dye Terminator Cycle Sequencing Ready Reaction Kit (PE Applied Biosystems). For restriction analysis, a 269-bp fragment containing exon 6 of the human SLC29A3 gene was PCR-amplified using the forward primer 5’-CAAGGGTTCGGGCTCACTG-3’ and the reverse primer 5’-TCTGCTCTCTGTCCCCAAGT-3’. The PCR products were subsequently digested with the BanII restriction enzyme (Takara Biotechnology Co., Dalian, China) and analyzed on agarose gel.
All 6 exons of SLC29A3 were amplified by polymerase chain reaction (PCR) from the genomic DNA using Premix LA Taq (Takara Biotechnology Co., Dalian, China) and previously reported primers.[2 (link)] The amplified PCR products were directly sequenced on an ABI Prism 3730×l Automated Sequencer, using the ABI Prism Big Dye Terminator Cycle Sequencing Ready Reaction Kit (PE Applied Biosystems). For restriction analysis, a 269-bp fragment containing exon 6 of the human SLC29A3 gene was PCR-amplified using the forward primer 5’-CAAGGGTTCGGGCTCACTG-3’ and the reverse primer 5’-TCTGCTCTCTGTCCCCAAGT-3’. The PCR products were subsequently digested with the BanII restriction enzyme (Takara Biotechnology Co., Dalian, China) and analyzed on agarose gel.
4
16S rRNA Gene Amplicon Sequencing
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Amplicons of 16S rRNA gene were sequenced using Y1 primer. DNA sequences were obtained with ABI PRISM Big Dye Terminator cycle sequencing-ready reaction kit (Applied Biosystems) using ABI 3100 (Perkin Elmer) capillary sequencer, following the manufacturer's instructions. The sequences used for the analyses were those with quality Phrep ≥ 20 [18 (link)]. A search for chimeric sequences was performed with Decipher software [19 (link)] and taxonomic identification to the level of phylum, class, order, family, and genus was made with the Classifier software using confidence limits of 80, 85, 91, 92, and 95% [11 (link)], respectively. Sequences that scored 95% confidence in the Classifier were used to search for similar sequences in MegaBlast (16S rRNA gene of the Bacterial and Archaea) [20 (link)] from the National Center for Biotechnology Information (NCBI) or the SeqMatch Ribosomal Database Project (RDP II) [21 (link)]. These searches were conducted in March 2016.
5
Bovine MHC Class II Genotyping
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BoLA-DRB3 alleles were genotyped using PCR-SBT. Briefly, exon 2 of BoLA-DRB3 was amplified by PCR as described by Takeshima et al. [19 (link)], using primers DRB3FRW and DRB3REV [28 (link)]. The PCR fragments were purified using an ExoSAP-IT PCR Product Purification Kit (USB Corp., Cleveland, OH) and sequenced using the ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Foster City, CA). Raw sequence data were analyzed using Assign 400ATF ver. 1.0.2.41 software (Conexio Genomics, Fremantle, Australia).
6
Bacterial 16S rRNA Gene Sequencing
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Bacterial clones were partially sequenced with use of the primer 27F. Sequence analysis involved use of an ABI PRISM Big Dye Terminator cycle sequencing-ready reaction kit and an ABI 3730 Genetic Analyzer (both Applied Biosystems, Foster City, CA, USA). Sequences were analyzed by use of the Mallard and Pintail programs to test for chimeras (Ashelford et al. [2005 (link), 2006 (link)]). Sequences were taxonomically assigned by use of the naïve Bayesian rRNA classifier, with confidence threshold 80% (Wang et al. [2007 (link)]) in the Ribosomal Database Project (http://rdp.cme.msu.edu/index.jsp ). The sequences have been deposited into GenBank under accession numbers JN168158-JN168654.
7
Bacterial 16S rRNA Gene Amplification and Cloning
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Total community DNA was extracted from 0.25 g of sediment with an PowerSoilTM DNA Isolation Kit (MoBio, Karlsbad, CA) according to the manufaturer’s instructions. Bacterial 16S rRNA gene fragments (~1500 bp) were amplified using PCR with the universal primer pair 27F(AGA GTT TGA TCC TGG CTC AG) and 1492R(AAG GAG GTG ATC CAG CCG CA) (Massol-Deya et al., 1995 ). The steps in PCR include initial denaturation at 94 °C for 5 min and 30 cycles consisting of denaturation at 94 °C for 1 min, primer annealing at 55 °C for 1 min, and extension at 72 °C for 1 min. The final elongation step was extended to 10 min. The resulting 1.5 kb PCR product was purified and cloned into pCR2.1-TOPO cloning vector and transformed into OneShot®DH5™ TOP10 chemically competent Escherichia coli cells according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA). Randomly selected clones were sequenced using an ABI PRISM Big Dye Terminator Cycle-Sequencing Ready Reaction kit (PE-Applied Biosystems, Foster, CA) and an automatic sequence analyzer (ABI 3700).
8
DNA Amplicon Purification and Sequencing
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The PCR amplicons were purified using the Wizard® SV Gel and PCR Clean-Up kit (Promega, Madison, WI, USA) following the instructions made available by the manufacturer. Nucleotide (nt) sequences were determined using the ABI Prism Big Dye Terminator Cycle Sequencing Ready Reaction Kit on an ABI 3730XL Genetic Analyzer (Applied Biosystems, Foster City, CA, USA).
9
Phylogenetic Analysis of Chlamydiae Using 16S rRNA Sequencing
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Sequencing of purified PCR products and plasmids was done using an ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction kit, v3.1 (Applied Biosystems, Perkin-Elmer) according to producer’s recommendations. Sequencing was done in both directions, and sequences used for phylogenetic studies originated from direct sequencing of PCR products. Sequencing was performed at the sequencing facility at the University of Bergen (http://www.seqlab.uib.no ).
An alignment of 56 16s rRNA gene sequences from the phylum Chlamydiae, retrieved from the GenBank or obtained from the present study, was made using Vector NTI 9.0 software. The alignment included members from all families within order Chlamydiales and several 16S rRNA gene sequences obtained from fish gills. Phylogenetic analysis was performed using TREE-PUZZLE 5.2 (available at:http://www.tree-puzzle.de ), maximum likelihood (ML). The best-fit nucleotide substitution model for the dataset was GTR+I+G, identified by Modeltest 3.6 (Posada and Crandall 1998 (link)). This model was implemented. Trees were viewed using TreeView (Page 1996 (link)).
An alignment of 56 16s rRNA gene sequences from the phylum Chlamydiae, retrieved from the GenBank or obtained from the present study, was made using Vector NTI 9.0 software. The alignment included members from all families within order Chlamydiales and several 16S rRNA gene sequences obtained from fish gills. Phylogenetic analysis was performed using TREE-PUZZLE 5.2 (available at:
10
Molecular Identification of Sarcocystis Species
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PCR products amplified through the use of the genus-specific primer set in the absence of the specific fragment for S. hirsuta, S. cruzi, S. hominis or S. bovifelis were sequenced to achieve species identification. PCR amplicons were purified with Exo-Sap treatment (USB Europe, Staufen, Germany) according to the manufacturer's instructions. Forward and reverse sequencing reactions were performed using ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction Kit, version 1.1 (Applied Biosystems, Foster City, CA). Sequenced fragments were purified by DyeEX (Qiagen, Hilden, Germany), and sequence analysis was performed on an Applied Biosystems 310 Genetic Analyzer (Applied Biosystems, Foster City, CA). The nucleotide sequences were analyzed using the BLASTN sequence similarity search at the NCBI database [18 (link)]. Phylogenetic analyses of the 18S rDNA gene sequences were performed using the neighbor-joining method [19 (link)] within MEGA7 [20 (link)]. Sarcocystis spp. reference sequences included are shown in Additional file 1 .
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