Question

Evaluate the statements below, and select those that correctly apply to the process of high throughput genome sequencing. Check All That Apply In the 4th step, the sequenced fragments are aligned, and the entire genome is computed using bioinformatics software. In the 4th step, the sequenced fragments are aligned, and the entire genome is computed using bioinformatics software. In the third step, the amplified fragments are read and sequenced based on the Sanger method. In the third step, the amplified fragments are read and sequenced based on the Sanger method. In the second step, the fragments bind to static probes and are amplified by PCR. In the second step, the fragments bind to static probes and are amplified by PCR. In the third step, gaps between contigs must be resolved to obtain the entire sequence. In the third step, gaps between contigs must be resolved to obtain the entire sequence. In the second step, each of the DNA fragments is engineered into a plasmid, and the plasmid is inserted into an E. coli cell to produce a library. In the second step, each of the DNA fragments is engineered into a plasmid, and the plasmid is inserted into an E. coli cell to produce a library. In the first step, the DNA is fragmented and fitted with short DNA sequences designed to match probes in the second step.

Answer 1

The exact steps based on the given options are as follows:

• In the first step, the DNA is fragmented and fitted with short DNA sequences designed to match probes in the second step.
• In the second step, the fragments bind to static probes and are amplified by PCR.
• In the third step, the amplified fragments are read and sequenced based on the Sanger method.
• In the 4th step, the sequenced fragments are aligned, and the entire genome is computed using bioinformatics software.

The above steps are followed in high throughput sequencing of parallel DNA using microbead technique. Here the DNA is flanked by an adapter, as mentioned in step 1 and then separated into single fragments. one of the single fragment is considered for binding to probes attached on the microbeads. These probes are complementary to one of the adapters with which the DNA sequence has been flanked. Therefore DNA single strand bind to the probe and forms a complimentary strand. when the reaction is complete one of the stand remains and the other is separated out to bind to a new probe. in this manner, a PCR reaction is executed inside the tube containing microbeads, where each bead with a DNA strand makes its own space for reaction to be carried out, owing to the oil-buffer interaction. On completion of the process, each strand is subjected to sequencing, either Sanger's or higher or newer methods like Pyrosequencing etc. The bases on each segmented strand is identified and are then assessed for overlapping sequences, based on which the software determines the whole genome sequence.

The two steps from the given options that do not place well in the technique are:

• In the third step, gaps between contigs must be resolved to obtain the entire sequence.
• In the second step, each of the DNA fragments is engineered into a plasmid, and the plasmid is inserted into an E. coli cell to produce a library.