Cycle sequencing is a variant of standard dideoxy, chain terminator DNA sequencing 1—3. The protocol has gained popularity owing to at least three features: simple execution, robust performance, and signal amplification. The main characteristic. The main characteristic that defines the cycle sequencing protocol is that the sequencing reactions are incubated in a thermal cycler, with programs similar to those used in polymerase chain reaction PCR.
This method assures efficient, reproducible utilization of even difficult templates by repeated thermal denaturation of the DNA template during the sequencing reactions. In fact, through heat cycling the same template molecules are used repeatedly, resulting in accumulation of sufficient sequencing signal even when a very limited amount of template is used, for example, when sequencing DNA from single bacterial colonies or phage plaques 4,5. It should be made clear, however, that no new template molecules are created as they are in PCR; cycle sequencing products accumulate linearly, not geometrically, in this single-primer DNA synthesis reaction.
Cycle sequencing can be performed using a variety of labeling and detection techniques. The following protocol utilizes radioactive end-labeled primer and X-ray film.
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Full text PDF Related articles. Abstract Cycle sequencing is a variant of standard dideoxy, chain terminator DNA sequencing 1—3. Related articles Based on techniques. MacBeath , , Springer Protocols See more. MacBeath et al.
Scofield et al. Associated articles This version , Robert W. Blakesley, References Murray, V. Nucleic Acids Res.
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Craxton, M. Methods Comp. Methods Enzymol. The amount of E. The estimated amounts of E. We set about evaluating the performance of the AMCC chip with human genomic DNA, a more complex genomic sample consisting of more than 3 billion nucleotide bases.
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Illumina MiSeq sequencing was selected as its throughput on the 2 by bp kit was higher than that achieved on the Ion Torrent PGM running a chip [ 18 ]. Fragmented genomic DNA from U-2 OS osteosarcoma cells ng was loaded into 14 of the 16 wells on the chip without prior size selection. Illumina MiSeq pair-end adapters were ligated and subsequently size-selected for bp.
For comparison, sequencing libraries were prepared off-chip manually using the standard protocols recommended by Ion Torrent using the IonXpress fragment library kit, Life Technologies, USA and Illumina using reagents from New England Biolabs , starting with ng of sheared genomic DNA.
Raw sequence data in fastq format were aligned to the genome of E. For Illumina sequence data, local alignment was performed in the paired-end mode --local. The protocols for preparing sequencing libraries from fragmented genomic DNA often include enzymatic reactions to generate blunt DNA ends, add 3'-dA tails to prevent concatemerization of genomic fragments during ligation, and ligation of dsDNA adaptors specific to the sequencing platform. Purification of the DNA is typically required after each enzymatic step.
The general workflow for preparing sequencing libraries on the AMCC chip was as follows: Fragmented genomic DNA was loaded into the sample inlet wells without prior size selection. The concentrated DNA on the column is then eluted into the sample chamber of the reaction circuit for the first automated reaction mix.
An important aspect of the device is that for each sample that is carried through the process, the purification and reaction circuits dedicated to that sample are each reused three times. Each time the sample DNA is eluted from the column, the binding capacity of the ChargeSwitch beads or carboxylated beads is regenerated by washing the column with fresh binding buffer. In addition, the binding buffer chamber is refilled with binding buffer.
By-products from each reaction are then removed via a waste outlet downstream of the column. For sample recovery, DNA from every reactor module is eluted into the collection wells and directly pipetted out. We assessed the efficiency of the NGS library preparation reactions performed on the AMCC chip by estimating the percentage of DNA fragments in the library with platform-specific sequencing adapters ligated on both ends.
The absolute amount of E. The absolute amount of DNA fragments with Illumina adapters ligated on both ends present in the library was estimated using RT-qPCR and a primer pair targeting the sequencing adapters.
To calculate DNA concentrations based on the RT-qPCR data, standard curves were generated for each primer pair using serial dilutions of a known amount of an Illumina sequencing library prepared from E. Reactors 8 and 16 were loaded with buffer as no template controls; no leakage of samples was observed from the neighboring reactor modules.
To evaluate the quality of the sequencing libraries prepared on the AMCC chip, we arbitrarily selected two Ion Torrent libraries and one Illumina library for sequencing. In parallel, we sequenced the control libraries that we had prepared using the conventional benchtop protocols recommended by the manufacturers. The throughput for the PGM sequencing runs ranged from 18 to 63 megabases per library, with the variation largely attributable to the differential loading of the ion sphere particles onto the Ion sequencing chip.
An average of 0. The MiSeq runs generated a higher throughput ranging from 50 to megabases, based on 2 to 11 million reads with a read length of 25 bp. The alignment rates for the reads was similar on both platforms. Mean sequencing depth for reactors 1 and 12 were 3. The median sequencing depths for all experimental runs as shown on Table 1 were close to the respective mean values, indicating that the distribution of the reads was not skewed in anyway.
The distributions of coverage depth were a close fit to the theoretical Poisson distribution for all cases Figure 3A. We estimated the library complexity for both manual and AMCC prepared libraries by randomly sub-sampling 10, reads from each run and determining the fraction of unique reads Table 1. No significant differences were observed with libraries generated using either method. We repeated the sub-sampling analysis using sample sizes ranging from 20, to , and still observed similar fractions of unique reads comparing the library prepared on-chip to those prepared using the benchtop methods Table S2 in File S1.
The run was successful, generating Average sequencing depth was calculated at 7. Alignment rate of the reads remained high at These results demonstrated the efficacy of on-chip library preparation for Ion Torrent sequencing. Table 1. Summary of sequencing data for the libraries prepared from E. The E. Libraries labeled "Control" were prepared off-chip using the standard benchtop protocols recommended by each manufacturer.
Sequencing runs with uniform coverage are expected to yield a Poisson distribution of coverage depths, indicated by the curves labeled "Theoretical limit". With the Illumina libraries that we prepared on the AMCC chip, the results from on chip preparation outperformed the control run in every aspect. With a higher throughput and hence 5 fold more number of reads, sequencing depth and genome coverage were significantly higher for the on chip library prepared. Alignment rates for the reads of the control and experimental runs were We generated an average In comparison, the control run had an average The uniformity of the runs was also confirmed with the distribution of coverage depth close to the theoretical Poisson limit as shown in Figure 3B albeit not as strongly fitted as the PGM runs.
Average sequence quality per base for the MiSeq data remained high for all 25 bases of each read Figure S6B in File S1 for both the control and on chip libraries.