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32

Think about it like this. Suppose you own a hundred copies of "The Lord of the Rings", a 500000 word novel. Unfortunately, you have those hundred copies in the form of several million tiny scraps of paper, each of which contains about ten sequential words from the novel. Your task is to take those several million scraps of paper and put them in order so that ...


29

Computers are used in several steps of sequencing, from the raw data to finished sequence (or not): Image processing Modern sequencers usually use fluorescent labelling of DNA fragments in solution. The fluorescence encodes the different base types. To achieve high throughput, millions or billions of sequencing reactions are performed in parallel in ...


21

The short answer is that corn genome is large and has a huge amount of duplication events. Around 80% of the genome are repeated. It's hard to assemble genomes with large amount of duplications because our sequencing technology, practically, at best can give ~500 base pairs. Figuring out the ordering of duplicated regions relies on scaffold sequences or ...


10

Here is a short summary of the sequencing technologies you listed. Illumina is the most frequently used one. Roche/454 FLX Pyrosequencer technology is based on pyrosequencing method, which utilizes the use of the enzymes ATP sulfurylase and luciferase. After the incorporation of each nucleotide by DNA polymerase, a pyrophosphate is released, which further ...


10

IMPORTANT EDIT : In your particular case, if you are working with bacterial genes, splicing is not an issue since bacteria do not have introns. I am leaving the information here since it may be useful to someone else. However, I recommend you focus on the UTRs since they are probably what is causing you problems. There are three things that could be ...


9

Perhaps you can draw inspiration from classic paper on lambda cloning: Maniatis T, Hardison RC, Lacy E, Lauer J, O’Connell C, Quon D, Sim GK, Efstratiadis A. 1978. The isolation of structural genes from libraries of eucaryotic DNA. Cell 15: 687–701. Try selecting tissues from the animal which you think is "enriched" (i.e. highly expressed) for the specific ...


9

Typically sanger sequencing will run into a few errors. Sometimes the traces will overlap as below in red and the computer will call N. If you truly wanted to figure out the correct basepair, you can look at the trace.


9

I think this is due to the over-representation of recognition sites with length 6: data<-c(16, 16, 12, 12, 6, 6, 6, 6, 4, 16, 6, 6, 6, 6, 15, 15, 6, 6, 6, 6, 11, 11, 6, 6, 4, 4, 6, 6, 11, 12, 6, 6, 23, 23, 6, 6, 6, 6, 9, 12, 4, 4, 6, 6, 6, 6, 6, 6, 6, 6, 6, 7, 7, 10, 10, 6, 4, 6, 6, 11, 11, 9, 9, 6, 6, 6, 6, 5, 5, 8, 8, 6, 6, 8, 8, 6, 9, 10, 10, 6, 6, 6, ...


9

Short Answer In a nutshell, DNA sequencing technology has a limit to how long a stretch of DNA it can read in one go. Long Answer So what most commonly occurs is the length of DNA you wish to sequence needs to be (almost randomly) chopped up into given lengths (depending on the technology) and each length or read is sequenced in parallel. But now you ...


8

This difference would have the greatest impact on treatment for cancer, in which a treatment protocol is based on genes deleted, amplified, altered in the tumor vs the reference genome for that patient. In terms of health risks based on SNP genotypes, the data are far from complete. Sure, some level of risk can be assigned to a variant (SNP), say at certain ...


8

The sequencing technologies that were developed in the last 20 years have a range of optimal use at an average A+T/G+C rate. Both highly AT-rich and GC-rich regions are complicated to process by the different sequencing technologies. Each technology has different ranges of usage, but to name one, Illumina technology prefers sequences in the middle range. If ...


8

All sequencing methods, be it classical Sanger sequencing or next-generation sequencing (or even third generation) need a certain amount of DNA to work with. You either need to extract DNA from a large-ish tissue sample or you need to amplify DNA content from a smaller sample. The first approach is often impractical, or downright impossible (when you want ...


8

After reading the paper cited I think the logic goes like this: DNAse I will create free ends at accessible sites in the genome. However shearing during subsequent DNA isolation is also a source of free ends, and these represent noise in the analysis. You have to put in a lot of energy to shear DNA to very small fragments, so I infer that mild shearing ...


8

Every nucleotide sequence has six possible reading frames, because each codon (determining one amino acid) consists of a base triplet (3 frames), and there is a complementary strand which could be coding (3 reverse frames). To find the possible open reading frames contained in your sequence, you have to look for start codons. That is: ATG. But, as said the ...


8

These are sequencing gels (in the cases here even radioactive ones) - they are run a lot longer than ordinary agarose gels and they are made from polyacrylamide. Im my experience, the most likely cause for skewing of lanes (not only bands) are samples, which still contain too much salts from the PCR reactions. This can also happen to only a few bands as seen ...


8

There are several nucleotide sequence datbases available. One of the largest is the NCBI GenBank at http://www.ncbi.nlm.nih.gov/genbank/ You may use the search bar at the top to search for nucleotide sequences belonging to a certain organism or gene. For example, searching for "E coli" will give you the full genome sequences for several bacterial species, ...


8

Non-biologist here stepping in. @swbarnes2 has a good point pinning the fact that (approx) 3Giga nucleotides to display "on a wall" (as you state) even with a good projector is gonna be a hard task. You'll need several projectors and a hell of a big wall. (say you take the smallest readable police setting you'll have each letter take a space of 4*6 pixels ...


7

Take a look at the strategies used to sequence the wheat genome. Wheat is hexaploid. The project is described at http://www.wheatgenome.org/. For early work on the maize genome, we employed methyl filtration in order to reduce genome complexity and size - transposons are filtered out and genes + promoters and such remain. The gene sequences are different ...


7

Why was decoding the genome so significant? Because decoding the genome gives us a complete picture of the genetic makeup of an organism. What made it so difficult? The repetitive sequences as mentioned by other people is a main problem. Imaging you are trying to complete a big puzzle with millions of pieces. Each piece represents a sequencing reads that we ...


7

The MIT synthetic chemist Gobind Khorana won the 1968 Nobel Prize in Chemistry for his work which successfully was able to make chains of Ribonucleic acids. The chemistry was difficult at the time but he won the prize for making specific sequences of RNA bases which were then fed to cells, resulting in specific amino acid chains, which ultimately deciphered ...


7

The first determination of a recognition site for a restriction endonulease was reported in: Kelly & Smith (1970) A restriction enzyme from Hemophilus influenzae II. Base sequence of the recognition site. J Mol. Biol. 51: 393-409 The enzyme was then called endonuclease R, but is now known as HindII (or HincII). The method used was to cut DNA with ...


7

The Next-Gen sequencers cannot sequence a very long stretch of DNA with good reliability (~150 for the recent model- HiSeq2000; even less for older models such as GA (40), GA-II (70), GA-IIx (90)). For increasing the confidence in a certain hit, it was sequenced from both the ends. For example, if you have selected 500bp DNA fragment, then after ligating ...


6

Another list of simple human traits can be found on the Western Kentucky University site. I will ask also AB0 and Rh Blood Groups, you can use this also to check associations with other genotypes/phenotypes, see this stackexchange question If you are going to sequence also the mitochondrial DNA, you may want to reflect on questions to ask also about their ...


6

The question is not too broad, just involves a fair bit of work to both do the research and compose a response. I'll do the latter, but in brief. The strings of the four nucleotides encode genes. Sometimes these genes are broken into protein-coding portions (exons) and sometimes intervening, non-coding regions (introns). Bacteria, for example do not have ...


6

In a genome, there are usually billions of base pairs. However, it's impossible to read all of them in one go. The DNA is fragmented, and the sequence of the fragments is determined. Next-generation sequencing techniques are faster and cheaper, but produce only short fragments (say, 100 base pairs, this depends on the technology). It's extremely ...


6

It's just a "string" where nucleotides encode something but I have no idea what they encode specifically. It sounds like you are describing a computer program, represented by a string of bytes on the hard drive. Unfortunately, the analogy breaks down very quickly because DNA is vastly more complex and a lot of aspects are still poorly understood. But ...


6

I can't comment on how A+T richness complicates the sequencing process itself, but I can comment on complications that arise when annotating the sequence. Ab initio gene predictors are often based on hidden Markov models that are very sensitive to base composition in the genome (di-nucleotides, tri-nucleotides, etc). These gene finders typically perform very ...


6

Note that the query sequence you have provided matches the minus strand of your target sequence. That means not only that the target sequence will be reversed (as you have noted), but it will also be complemented as well. So in the GenBank record, you should not look for the sequence CCGACCGA... starting at position 10835, you should look for the sequence ...


6

I'm assuming you mean DNA sequencing (excluding things like RNA-seq). Is Sanger sequencing the first generation? From Metzker 2010: The automated Sanger method is considered as a "first-generation" technology Some of the technology that was in development when this review was written is no longer in development, but this is still an excellent review ...



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