I previously read this question and this paper and learned good things about this topic. my current question is that scientists use which tools, algorithms, soft wares... to sequence, assemble and annotate plant genomes? For each genome project do they develop new tools and algorithms or there are some tools that are useful for (almost) all genome projects? Are there any comprehensive site, book or paper that have plant genome sequencing and annotation protocols?

  • $\begingroup$ Given the C-value paradox (where there may be massive amounts of non-coding DNA, much of it often repetitive), and the fact that polyploidy seems more frequent in the plant kingdom than in animals, (e.g., bananas, wheat), I can imagine some challenges at the assembly step, but I am not aware of a specific tool or approach that is only used by plant genome projects. DNA is DNA for every kingdom, and eukaryotic genes are eukaryotic genes, regardless of species, right? Software and algorithms don't care where the DNA comes from. $\endgroup$ – mdperry Jun 10 '15 at 11:54
  • $\begingroup$ But I think algorithms will consider long repeats in plant genomes $\endgroup$ – MySky Jun 10 '15 at 12:12

Let's try and answer all three parts of your question.

Sequencing 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 genomes. To get long reads, you need long input DNA sequences. Therefor you would want to follow a suitable protocol for high molecular weight plant DNA. That might be hard, because plant DNA can be difficult to extract based on the plant and tissue you have, as most easily put you have to "crack open" the cell wall in the very beginning. After that, it is general sequencing. Although, as I already said you might opt for long reads (PacBio) and/or good coverage. If that is not at all feasible, you might choose to do targeted sequencing and only capture the whole exome (or only the genes you are interested in) to reduce both cost and analysis complexity.

Assembly Now you have probably accumulated a lot of sequence and also potentially long sequence. There are multiple tools you could use for assembly and generally you can use the same for plants that you might want to use for example for a human genome assembly. One example that works well on large plant genomes is MaSuRCA, another is Allpaths-LG. There are some assemblers designed to cope with long repeats, but quite often you just assemble and then try figuring out the repeat regions later, for example by improving a shotgun read assembly with long reads from another technique.

Annotation This one definitely has specific tools for plant genomes. Well, large genomes. The main thing in annotation is finding potential genes and comparing them to databases to annotate them with function. Plants do splice and plants can have long introns, even ones including long repeats. One gene prediction software example that works on plants is Augustus, but for some model organisms there are even organism-specific pipelines and tools for this task. And of course, on the database part you would choose to rely on a plant specific database (if you do not use NCBI-NR anyway). Those can also be species or genus specific like the ones you find on the TAIR (Arapidopsis) project.

TLDR: It's in fact complicated. On one hand, there is a need of specific tools and databases, on the other you might go well with standard tools, but would have to adapt parameters, do additional pre- or post-processing steps and so on.

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  • $\begingroup$ Thank you. very use full answer. I have a question: in annotation part, if I have a set of assembled sequences, how can I compare (align?) all them with databases like NCBI or TAIR to find possible matches? Are there automatic ways or I'll code or do that manually? $\endgroup$ – MySky Jun 10 '15 at 16:27
  • $\begingroup$ There is a variety of alignment tools, the most used and most widely known is BLAST. There is a web server at blast.ncbi.nlm.nih.gov/Blast.cgi but for complete plant assemblies or even all proteins found in one plant you would have to install and run it locally. In case you are not aligning pre-predicted protein sequences but want to align against RNA or protein databases, you would need a spliced aligner. $\endgroup$ – skymningen Jun 11 '15 at 7:04

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