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--- assemblies:2015:mitochondrion_assembly [2015/07/23 04:43]
ndudek [PCR results]
+++ assemblies:2015:mitochondrion_assembly [2015/09/16 01:30] (current)
ndudek [PCR results]
@@ Line 33: / Line 33: @@
 |mitochondrion_iteration2_SW018-9_discovar| 48K | 45,494 | 114 | 9,030 | 9,030 | 15,548 | 0 | 436X |
-For comparison, the mitochondrion size of the closest related molluscs that have mitochondrion assemblies are 14,100bp (grove snail - //Cepaea nemoralis//) and 14,130bp (land snail - //Albinaria coerulea//)
+For comparison, the mitochondrion size of the closest related molluscs that have mitochondrion assemblies are 14,100bp (grove snail - //Cepaea nemoralis//) and 14,130bp (land snail - //Albinaria coerulea//). But there are mollusk mitochondrial genomes that are much larger: sea scallop //Plactopecten magellanicus// is reported to have 30.6-30.7kbp ([[http://link.springer.com/article/10.1007/s00239-007-9016-x#page-1|David R. Smith and Marlene Snyder. Complete Mitochondrial DNA Sequence of the Scallop Placopecten magellanicus: Evidence of Transposition Leading to an Uncharacteristically Large Mitochondrial Genome. J Mol Evol (2007) 65:380–391 doi:10.1007/s00239-007-9016-x]]).
 It looks as though the majority of the mitochondrion is in two contigs in the assembly using both the SW018 and SW019 assemblies. One of these contigs is 12,884bp and the other (which is the second largest contig) is 3,425bp.
@@ Line 204: / Line 205: @@
   - Some kind of technical error or PCR artifact.
   - The smaller contig may be a nump that, for some reason, circularized. Supporting this theory (possibly) is     that if you blast the larger contig against the //Albinaria caerulea// genome, it looks like we have nearly the entire mitochondrial genome, expect for an ~800bp region (see dot plot below). This is only slightly shorter than the fragment amplified with the PCR above. Given that molluscs seem to have fairly conserved mitochondrial genome sizes of ~14,000bp, if both fragments are truly mitochondrial it means the total length must be over 16,309bp, which is unexpectedly large and may be incorrect. On the other hand, if the second largest contig is a nump, you would likely expect it to have some sequence similarity to the largest contig, but blasting them against each other finds no significant sequence similarity. Additionally, I used [[http://dogma.ccbb.utexas.edu/|DOGMA]] to do a preliminary annotation of both contigs, and it predicted that the largest carries the cox1 and cox 3 genes, whereas the second largest contig carries the cox2 and cob genes, so they appear to be complimentary.
-  - The banana slug could possibly have a mitochondrial genome that is present as separate mini circular chromosomes, as seen in the human body louse, //Pediculus humanus// (see "The single mitochondrial chromosome typical of animals has evolved into 18 mini chromosomes in the human body louse, //Pediculus humanus//", by Shao //et al//., 2009). This could explain why both contigs circularized with the PCR that was run. However, it is probably too early to say whether this circularization is really "valid" or not. This is something that will be examined in greater detail in the future.
+  - The banana slug could possibly have a mitochondrial genome that is present as separate mini circular chromosomes, as seen in the human body louse, //Pediculus humanus// (see [[http://genome.cshlp.org/content/19/5/904.short|"The single mitochondrial chromosome typical of animals has evolved into 18 mini chromosomes in the human body louse, Pediculus humanus", by Shao et al., doi:10.1101/gr.083188.108 Genome Research 2009. 19: 904-912]]). This could explain why both contigs circularized with the PCR that was run. However, it is probably too early to say whether this circularization is really "valid" or not. This is something that will be examined in greater detail in the future. Note: [[http://genome.cshlp.org/content/19/5/700.full|‘Why genomes in pieces?’ revisited: Sucking lice do their own thing in mtDNA circle game. David M. Rand,
+Genome Research. 2009. 19: 700-702. doi:10.1101/gr.091132.109]] lists other animals with multi-chromosome mitochondria, including a mollusk (scallop).
 {{:assemblies:2015:longest_vs_a_caerulea.png?200|}}
+August 2015
+PCR products were sent for Sanger sequencing. Something went wrong - the result was largely "N"s. I checked whether I could improve calls by looking at the chromatogram, but did not have significant success. Below is an example of what the chromatogram looked like. I will be re-sending the PCR products for sequencing.
+{{:assemblies:2015:chromatogram_mito_august2015.png?200|}}
+September 2015
+PCR products were sent for sequencing a second time, this time after a size selection on the strongest band. Results were essentially the same.
 The mitochondrion assembly is being worked on by Natasha Dudek (natasha@dudek.org) from Team 5: Discovar //de novo//.

Banana Slug Genomics

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