Q: What are the advantages of next-generation sequencing technology over traditional Sanger sequencing?
A: Sanger sequencing took 13 years to complete the first human genome map, and today the next-generation sequencing technology platform can be completed in just a few months. The next-generation sequencing technology platform uses a large number of parallel processing capabilities to read multiple short DNA fragments and then stitch them into longer contig and scaffold. The technology is more advanced, with advantages of high accuracy, high throughput, high sensitivity and low operating cost.
Q: What do contig, scaffold, N50, etc. represent from scratch?
A: Read: the length of the nucleotide read by the instrument in one sequencing
Contig: A unit formed by assembling adjacent reads by overlapping portions is called contig.
Scaffold: Using information from other methods such as double-end sequencing, locates the linear or relative positional relationship of contigs on chromosomes and joins them to form longer scaffold sequences.
N50: Sorts contig or scaffold from large to small and accumulates their length. When the accumulated length reaches half the length of the genome sequence, the last contig or scaffold length.
Q: What is the frame picture, the fine picture, and the completed picture in the de novo antibody sequencing?
A: The frame diagram refers to the genome coverage greater than 95%, the coverage of the gene region is over 98%, the contig N50 reaches 5Kb, the scaffold N50 reaches 20Kb, and the single base error rate is 100,000. One or less.
The fine map refers to the genome coverage greater than 98%, the coverage of the gene region is over 99%, the contig N50 reaches 20Kb, the scaffold N50 reaches 300Kb, and the single base error rate is less than 100,000 points after bioinformatics analysis. One of them, the number of gaps does not exceed 100.
The completion map refers to the complete genomic sequence obtained by bioinformatics analysis, and the single base error rate is less than one in 100,000.
Q: What are the requirements for whole-genome sequencing for DNA samples?
A: The OD value of DNA should be between 1.8 and 2.0; the higher the concentration of the sample, the better, the minimum should not be less than 200 ng / ul; the total amount of sample DNA is not less than 10 ug.
Q: What are the requirements for sampling the sample by animal genome sequencing?
A: Samples should be extracted from areas with low fat content such as muscle and blood. Try to use the same individual to sample. If the species size is small, the amount of DNA extracted by a single body is less than one sequencing reaction. In the case of guaranteed amount, the number of species should be minimized to reduce the influence of individual differences on subsequent splicing.
A: (1) Both sequencing methods can complete the De Novo protein sequencing splicing of the genome. It is recommended to use two sequencing methods, and the results can be combined and spliced to reuse the advantages of the two technologies.
(2) According to the situation of the genome, if the heterozygosity is high and the repeat sequence is too high, it is recommended to increase the proportion of 454 sequencing.
(3) If the genome is relatively simple, it is recommended to increase the Solexa sequencing ratio from the cost considerations.
(4) 454 is mainly a cost and flux limitation. Solexa is mainly unable to overcome short tandem repeats above 150 bp.
(5) 454 can't test short tandem repeats above 700bp. If this level of repeats causes a splicing bottleneck, Sanger data needs to be added.
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