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 ...
There are multiple ways of doing genome assembly.
The term you are probably looking for is "De Bruijn-Graph based assembly". Using this you can find a lot more different explanations of how it is done.
Another frequently used method is "Overlap Layout Consensus assembly", which in fact is not based on k-mer counting.
Let's try and answer all three parts of your question.
The general method is the same. Sequencing is just sequencing. But as for every single sequencing, there are factors to consider and protocols to be selected.
One important thing is, that you might want comparably long reads to cope with the repeats and the general large size of plant ...
It is done for checking sequence similarity between two or more different sequences. This will give information about how two sequences are different, what is their evolutionary relationship, which residues are conserved etc. Take a look at following sequence alignment between different sequences. (Image courtesy: Wikimedia Commons)
You can try looking around biostars.org, which is like stackexchange, but for bioinformatics.
Velvet is one example of a de novo assembler.
But 30 bp is really short, and animals have big genomes (not as tough as lots of plants and fungi, but still tough)
What you would get is a bazillion short contigs. It would not be pretty.
In Illumina sequencing, the DNA is (usually randomly) sheared into fragments. For paired end sequencing, fragments of a specific size range are selected and then sequenced from both sides.
This results in two reads for each fragment. As read length is fixed, also the remaining "middle part" of the fragment is in a specific size range. In some cases there is ...
I suspect mispriming due to annealing temp being too low compared to predicted melting temp of overlap regions. As the protocol in your link notes:
(3) Choose annealing temperature wisely. We recommend to use the same
as min_Tm by Primerize design, which is usually between 60-64 °C.
(4) Check PCR product on 4% agarose gel. If assembly is unsuccessful
Generally, gaps in genomes occur due to failure of de novo assembly -- the DNA can be sequenced accurately, but cannot subsequently be placed into a contiguous assembly due to alignment ambiguity with adjacent sequences. In eukaryotes, telomeres and centromeres are difficult to assemble from short reads because they are highly repetitive. In bacteria, ...
If you only want to use only sequencing techniques, you have a problem.
To get a feeling of what kind of results to expect, consider this paper published recently in Nature Genetics. They tried to assemble a whale genome de novo. They had 7 (!) paired-end libraries with different insert lengths ranging from 170bp to 20kb. Read lengths were mostly 100bp and ...
It means k-mer coverage. From the velvet manual:
4.2.1 The contigs.fa file
This fasta file contains the sequences of the contigs longer than 2 k
, where k is the word-length used in velveth. If you have specified a
min_contig_length threshold, then the contigs shorter than that value are omitted. Note that the length and coverage ...
If the DNA sequence contains repetative elements, and your reads are not long enough to resolve them, you won't know if you have the right path. Usually you split your results into contiguous regions whose sequence you are confident in.
The publication of the first complete DNA sequence of Escherichia coli was for strain K-12 substr. MG1655 in 1997. Because of historical limitations in the technology used (rather than problems with repeat sequences or instability in the genome) this contained mistakes, which were subsequently corrected as the sequencing technology improved. An ...
I'm going to re-interpret this question slightly to make it easier to answer:
"Why do genome assemblies frequently consist of large numbers of short contigs rather than a relatively small number of long chromosomes (or full replicons of other types)? And how could I make my assembly better?"
De novo sequence assemblies (usually) consist of a set of ...
One that isn't on your list: cost.
In the form of Oxford Nanopore data, we have extremely long reads of low accuracy. These are not too expensive but on their own they are probably not viable for many applications.
In the form of Illumina data, we have extremely abundant, cheap, high-accuracy short reads, suitable for "counting" approaches. This ...
I have never heard the term “contig based alignment”, and your question is the only Google hit of this exact query (apart from a 2012 patent application).
That said, and without knowing the exact context, I am assuming that you are essentially right: contig-based alignment probably refers to the de novo assembly of reads into contigs, which are then aligned ...
As clearly stated in the WP article:
The molecule is made up of 20 complement control protein (CCP) modules (also referred to as Short Consensus Repeats or sushi domains) connected to one another by short linkers (of between three and eight amino acid residues) and arranged in an extended head to tail fashion.
So in your terminology yes, it is a repeat ...
The two tasks are quite different.
Aligning means comparing sequences to find differences between them.
Assembling means that you take a bunch of short sequences and try to stitch them back together to reconstitute the original DNA sequence.
Fig1: Screen capture from DNA Baser Assembler of an 'assembly to reference'. The reference is in purple color. The ...
This is an old question but it seems relevant to answer now given current state of art. See also this related thread on biostars.
There are now many tools that perform this function, which is somewhat routine. A partial list:
RagTag (successor of RagOO)
I don't know which is best. Quickmerge is the one that I ...