EGFR exons 19 and 20 and KRAS exons 2 and 3 were amplified by PCR (for primer pairs see S1 Table ). Amplified products were then purified using Exostar 1-Step (VWR International) according to the manufacturer's instructions. Sequencing reactions were performed using the Big Dye Terminator version 3.1 (Applied Biosystems, Foster City, CA, USA). Dye purification was carried out by Centrisep Spin columns (Princeton Separation) and subsequent sequencing analysis was resolved on a 3130XL Genetic Analyzer (Applied Biosystems). Sequences were finally analyzed with Sequence Analysis v5.2 and SeqScape v2.5 (Applied Biosystems).
Sequence analysis v5
Manufactured by Thermo Fisher Scientific
Sourced in United States
Sequence Analysis v5.2 is a software tool for analyzing DNA and protein sequences. It provides a range of functions for sequence alignment, phylogenetic analysis, and visualization.
Automatically generated - may contain errors
Lab products found in correlation
Seqscape v2, by Thermo Fisher Scientific (1 mentions)
Bigdye terminator version 3, by Thermo Fisher Scientific (1 mentions)
3130xl genetic analyzer, by Thermo Fisher Scientific (1 mentions)
Nanodrop, by Thermo Fisher Scientific (1 mentions)
Ampure magnetic beads, by Beckman Coulter (1 mentions)
Sequencher, by Gene Codes (1 mentions)
Dneasy blood and tissue kit, by Qiagen (1 mentions)
Bigdye terminator mix v3, by Thermo Fisher Scientific (1 mentions)
Bigdye terminator v3, by Thermo Fisher Scientific (1 mentions)
Sequencher v4, by Gene Codes (1 mentions)
3730 dna analyzer, by Thermo Fisher Scientific (1 mentions)
4 protocols using sequence analysis v5
1
Targeted Genomic Sequencing of EGFR and KRAS
2
Molecular Identification of Species via Sequence Alignment
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Quality of sequences was determined using Sequence Analysis v5.2 software (Applied Biosystems, Foster City, CA). Apart from that CLUSTALW algorithm implemented in BioEdit version 7.0.5.3 (Hall 1999 ) was used in the multiple sequence alignments (MSA). The sequences obtained from the four different species were compared with sequences available on NCBI through a BLAST search (http://blast.ncbi.nlm.nih.gov/ ) (Table 1 ). For the species diagnosis, we considered the percentage similarity between query and reference sequence pairs. To confirm MSA result, we compare our data with phylogenetic analysis conducted using MEGA version 7.0 (Kumar et al. 2016 (link)) for 12S rRNA using the neighbor-joining (NJ) method (Saitou and Nei 1987 (link)). Estimates of evolutionary divergence over sequence pairs between groups determined by using the Kimura 2-parameter distance (Kimura 1980 (link)) as implemented in MEGA version 7.0 (Kumar et al. 2016 (link)).
3
BCL2 Gene Sequencing Protocol
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For the 14 cell lines, genomic DNA was extracted using the DNeasy blood and tissue kit (Qiagen). DNA was quantified using a Nanodrop spectrophotometer (Thermo Fisher, Waltham, MA). A total of four amplicons covering BCL2 exons 1 and 2 were sequenced using the primers and cycling conditions (Additional file 1 : Table S1). 10 μl of the PCR products were purified using AMPure magnetic beads (Beckman Coulter, Inc., Brea, CA) and eluted in 40 μl of sdH2O. 4 μl of the purified product was cycle sequenced using Big Dye Terminator Mix v3.1(Applied Biosystems, Grand Island, NY) at 1/16 chemistry. Both forward and reverse orientations of the PCR products were sequenced and applied to an Applied Biosystems 3130xl DNA Sequencer (Applied Biosystems, Grand Island, NY). Base calling was performed using Sequence Analysis v5.2 (Applied Biosystems, Grand Island, NY) with the KB basecaller v1.2. Sequence data was aligned to the reference BCL2 sequence (NM_000633.2) using Sequencher (Gene Codes Corp., Ann Arbor, MI).
4
DNA Sequencing Protocol for Chromosome 9
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DNA sequencing reactions were performed with both forward and reverse primers for each PCR amplicon using BigDye Terminator v3.0 chemistry (Applied Biosystems Inc.) utilizing the following thermal profile: 96°C for 6 min, then 25 cycles of 96°C for 10 s, 50°C for 5 s, 60°C for 4 min. Sequencing reaction purification was carried out using either ethanol precipitation or an Agencourt CleanSeq method (Beckman Coulter Inc., Danvers, MA, USA). Purified DNA sequencing reactions were electrophoresed on the Applied Biosystems Inc. 3730 DNA Analyzer, in the GaP Facility of the CREAIT Network at Memorial University of Newfoundland.
Raw data was collected using Sequence Analysis v5.2 (Applied Biosystems Inc.) and imported into Sequencher v4.8 (Gene Codes Corp., Ann Arbor, MI, USA). Contigs were created by assembling reads to the reference sequence, Accession Number NC_006591.3 (chromosome 9) using an 85% minimum gap percentage and a 20% minimum overlap, followed by manual trimming and editing of sequence each read.
Raw data was collected using Sequence Analysis v5.2 (Applied Biosystems Inc.) and imported into Sequencher v4.8 (Gene Codes Corp., Ann Arbor, MI, USA). Contigs were created by assembling reads to the reference sequence, Accession Number NC_006591.3 (chromosome 9) using an 85% minimum gap percentage and a 20% minimum overlap, followed by manual trimming and editing of sequence each read.
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