DNA sequencers that replaced the radioactive labels and autoradiography in Sanger’s method with fluorescent labels and laser-based detection (2). In Smith’s four-spectral- channel, single-lane sequencer, four fluorescently labeled primers are associated with the terminating dideoxynucleotide through use of separate chain-terminating reactions.Fluorescence, excited by two laser lines and detected in four spectral channels, is used to identify the terminal nucleotide.
In Ansorge’s single-spectral-channel, four-lane sequencer, a single fluorescent label is used with each dideoxynucleotide chain-terminating reaction and the products are run on separate
lanes of a slab gel; sequence identification is similar to the classic autoradiography technique. In Prober’s twospectral-channel, single-lane sequencer, one of four fluorophores is associated with the terminating dideoxynucleotide.
When double-stranded pUC-based subclones are used as templates, the amount of primer is doubled and a denaturing/annealing step is added. Here, 3 ug of plasmid DNA, isolated by either the mini- or midi-prep diatomaceous earth method, is mixed with primer, placed in a boiling-water bath, and rapidly cooled by plunging into an ethanol/dry-ice bath (28). Following an incubation on ice, the remaining sequencing extension reagents (reaction buffer, nucleotide extension mix.To denature the DNA and anneal the primer, incubate the following reagents in a boiling water bath for 4-5 minutes and rapidly cool the reaction by plunging into an ethanol/dry ice bath.
Each base-specific sequencing reaction terminated with the short termination mix is loaded using a mouth pipette onto a 0.15 mm X 50 cm X 20 cm, denaturing 5% polyacrylamide gel and electrophoresed for 2.25 hours at 22 mA. The reactions terminated with the long termination mix typically are divided in half and loaded onto two 0.15 mm X 70 cm X 20 cm denaturing 4% polyacrylamide gels. One gel is electrophoresed at 15 mA for 8-9 hours and the other is electrophoresed for 20-24 hours at 15 mA. After electrophoresis, the glass plates are separated and the gel is blotted to Whatman paper, covered with plastic wrap, dried by heating on a Hoefer vacuum gel drier, and exposed to X-ray film. Depending on the intensity of the signal and whether the radiolabel is 32-P or 35-S, exposure times varied from 4 hours to several days.
Double-stranded dye-terminator reactions required approximately 5 ug of diatomaceous earth modified-alkaline lysis midi-prep purified plasmid DNA. The double-stranded DNA is denatured by incubating the DNA in sodium hydroxide at 65degC, and after incubation, primer is added and the reaction is neutralized by adding an acid-buffer. Reaction buffer, alpha-thio-deoxynucleotides, fluorescent-labeled dye-terminators, and diluted Sequenase[TM] DNA polymerase then are added and the reaction is incubated at 37degC. Ammonium acetate is added to stop the reaction and the DNA fragments similarly are precipitated, rinsed, dried, and stored.
After data collection, an image file is created by the ABI software which related the fluorescent signal detected to the corresponding scan number. The software then determined the sample lane positions based on the signal intensities. After the lanes are tracked, the cross-section of data for each lane are extracted and processed by baseline subtraction, mobility calculation, spectral deconvolution, and time correction. On the Macintosh computer, the collected data can be viewed in several formats.
The FLX instrument currently provides 100 flows of each nucleotide during an 8-h run,which produces an average read length of 250 nucleotides (an average of 2.5 bases per flow are
incorporated). These raw reads are processed by the 454 analysis software and then screened
by various quality filters to remove poor-quality sequences, mixed sequences (more than one initial DNA fragment per bead), and sequences without the initiating TCGA sequence. The
resulting reads yield 100 Mb of quality data on average. Downstream of read processing, an assembly algorithm (Newbler) can assemble FLX reads. Although shorter than reads derived from capillary sequencers,FLXreads are of sufficient length to assemble small genomes such as bacterial and viral genomes to high quality and contiguity.As mentioned, the lack of a bacterial
cloning step in the Roche/454 process means that sequences not typically sampled in a WGS
approach owing to cloning bias will be more likely represented in a FLX data set, which con-
Bridge amplification
In Ansorge’s single-spectral-channel, four-lane sequencer, a single fluorescent label is used with each dideoxynucleotide chain-terminating reaction and the products are run on separate
lanes of a slab gel; sequence identification is similar to the classic autoradiography technique. In Prober’s twospectral-channel, single-lane sequencer, one of four fluorophores is associated with the terminating dideoxynucleotide.
When double-stranded pUC-based subclones are used as templates, the amount of primer is doubled and a denaturing/annealing step is added. Here, 3 ug of plasmid DNA, isolated by either the mini- or midi-prep diatomaceous earth method, is mixed with primer, placed in a boiling-water bath, and rapidly cooled by plunging into an ethanol/dry-ice bath (28). Following an incubation on ice, the remaining sequencing extension reagents (reaction buffer, nucleotide extension mix.To denature the DNA and anneal the primer, incubate the following reagents in a boiling water bath for 4-5 minutes and rapidly cool the reaction by plunging into an ethanol/dry ice bath.
Each base-specific sequencing reaction terminated with the short termination mix is loaded using a mouth pipette onto a 0.15 mm X 50 cm X 20 cm, denaturing 5% polyacrylamide gel and electrophoresed for 2.25 hours at 22 mA. The reactions terminated with the long termination mix typically are divided in half and loaded onto two 0.15 mm X 70 cm X 20 cm denaturing 4% polyacrylamide gels. One gel is electrophoresed at 15 mA for 8-9 hours and the other is electrophoresed for 20-24 hours at 15 mA. After electrophoresis, the glass plates are separated and the gel is blotted to Whatman paper, covered with plastic wrap, dried by heating on a Hoefer vacuum gel drier, and exposed to X-ray film. Depending on the intensity of the signal and whether the radiolabel is 32-P or 35-S, exposure times varied from 4 hours to several days.
Double-stranded dye-terminator reactions required approximately 5 ug of diatomaceous earth modified-alkaline lysis midi-prep purified plasmid DNA. The double-stranded DNA is denatured by incubating the DNA in sodium hydroxide at 65degC, and after incubation, primer is added and the reaction is neutralized by adding an acid-buffer. Reaction buffer, alpha-thio-deoxynucleotides, fluorescent-labeled dye-terminators, and diluted Sequenase[TM] DNA polymerase then are added and the reaction is incubated at 37degC. Ammonium acetate is added to stop the reaction and the DNA fragments similarly are precipitated, rinsed, dried, and stored.
After data collection, an image file is created by the ABI software which related the fluorescent signal detected to the corresponding scan number. The software then determined the sample lane positions based on the signal intensities. After the lanes are tracked, the cross-section of data for each lane are extracted and processed by baseline subtraction, mobility calculation, spectral deconvolution, and time correction. On the Macintosh computer, the collected data can be viewed in several formats.
The FLX instrument currently provides 100 flows of each nucleotide during an 8-h run,which produces an average read length of 250 nucleotides (an average of 2.5 bases per flow are
incorporated). These raw reads are processed by the 454 analysis software and then screened
by various quality filters to remove poor-quality sequences, mixed sequences (more than one initial DNA fragment per bead), and sequences without the initiating TCGA sequence. The
resulting reads yield 100 Mb of quality data on average. Downstream of read processing, an assembly algorithm (Newbler) can assemble FLX reads. Although shorter than reads derived from capillary sequencers,FLXreads are of sufficient length to assemble small genomes such as bacterial and viral genomes to high quality and contiguity.As mentioned, the lack of a bacterial
cloning step in the Roche/454 process means that sequences not typically sampled in a WGS
approach owing to cloning bias will be more likely represented in a FLX data set, which con-
Bridge amplification
