Molecular Biology...From Scratch?

The motto of my dissertation research has been "we're molecular biologists, we can do that" and we did often from scratch. My training took a turn for the silicon when I embarked on transcriptomics/genomics projects. I spend most of my time here describing technical troubleshooting and genomics methods.

[rustbeltscientist]

The use of base quality scores in genome alignment is quite variable, here is an example of that usage in Bowtie. The Bowtie and Bowtie2 manuals have a detailed description of these parameters.

[rustbeltscientist]

The changes to RNA-Seq library prep include:

1. The use of direct isolation of mRNA

The concern here is not knowing the integrity of the RNA isolated-but with cheaper sequencing and models to adjust for degradation it may not be a future issue. On the other hand this technique promises to reduce rRNA contamination.

2. Enzyme based fragmentation

3. Increased reliance on bead based purification. 

I will say that the difference between gel slice purification and Ampure XP is the difference between success and failure of library prep in my case. 

[scienceisbeauty]

How to Extract DNA from Anything Living (click).

Via Learn Genetics (The University of Utah)

[the-mighty-ribozyme]

Neat! Educators take note!

[molecularlifesciences]

Used this for outreach events! It is great!!!!

[rustbeltscientist]

I remember doing this as a kid, it obviously made some impression on me.

[rustbeltscientist]

A wonderful hour long interview with Irene Pepperberg at the Chicago Humanities Festival about her work with Alex the African Grey

[sciencenote]

The insertion of one gene can muzzle the extra copy of chromosome 21 that causes Down’s syndrome, according to a study published today in Nature. The method could help researchers to identify the cellular pathways behind the disorder’s symptoms, and to design targeted treatments.

Lawrence...

[whatshouldwecallgradschool]

UNSTICKING THE DOOR OF THE -80 FREEZER

credit: lilium

[rustbeltscientist]

Indel Realigner can improve assemblies!

Congratulations to the individual in my former lab who has been doing all the work on this. Several rounds of local realignment around indels using GATK. Informed by indel calling and generating fresh consensus sequences can improve alignments in some cases. Neat.

[rustbeltscientist]

http://users.ugent.be/~avierstr/nextgen/Next_generation_sequencing_web.pdf

One of the most up-to-date comparisons of Next Gen Sequencing technologies I have seen to date by Andy Vierstraete of Ghent University.  

[rustbeltscientist]

Find Next Generation Sequencing Centers by platform and location

[phytopathology-deactivated20170]

Conidial chains borne atop conidiophores.  It is these stalk-like conidiophores that give mildew colonies their powdery appearance.  Spores form at the bottom of the chain, so the oldest spores are at the tip of the chain.  They break off in wind currents and can travel short distances, generally less than 100 meters. (Photo and description by David Gadoury, Cornell University)

[columbiascience]

Breast Cancer Survival Predicted By Computer Model

by Rachael Rettner Published April 17, 2013

A new computer model may help researchers better predict how long breast cancer patients will live, a new study suggests.

The model, which was developed in a researchers’ contest, uses gene signatures — sets of genes that are all “turned on" at the same time in a patient’s cancer — to estimate how long patients will live.

Read More

[dailysciencefacts]

today's science fact is about an animal/plant fusion: Elysia chlorotica!

E. chlorotica is a sea slug that literally sucks the chloroplasts out of algae and preserves them to use for photosynthesis. Once a baby slug ingests enough chloroplasts it can potentially never have to eat again, relying on the sun for its energy needs. Recent studies have found that the slug encodes and expresses plant genes encoding chlorophyll- the green pigment used in photosynthesis- which needs to be continuously replaced in chloroplasts.

[rustbeltscientist]

Genomics resources

Reading up on who the Galaxy Project follows on twitter was enlightening

Evolution and Genomics workshops resources posted from the 2013 genomics workshops include intro and advanced unix, intro to python and R. The 2013 Molecular Evolution has PAUP, PAML and Maximum Likelihood. They can also be found on twitter as @evomics

[aperturesciencejournalclub-blog]

I think this is best described as "Project Euler, except more useful for aspiring bioinformaticians"!  This features a list of computational biological relevant problems, starting with the very basic ("for a given strand of DNA, return its complement") to moderate (assembling a (smallish!) genome) to difficult (identifying protein motifs).

As a biologist with aspirations to computational biology, this looks perfect for me.  H/t for the link goes to troisroyaumes, the computational biologist I look up to in life :)

[rustbeltscientist]

Well this looks really neat

[biochemistries]

In the first paper of its kind, publicly available genomic data has been used to examine the extent to which bacterial DNA is integrated into the human somatic genome by lateral gene transfer (LGT). Viral DNA is already known to find its way into our genomes, as well as to integrate into bacterial host DNA.

Given that the bacteria that colonise us outnumber our own cells 10 to 1, perhaps this should not come as a surprise. The group at the University of Maryland now believe that LGT occurs through an RNA intermediate, finding that such integrations are more frequent:

in tumours than in normal cells (210× more),

in (r)RNA than DNA, and

in the mitochondrial genome than the nuclear genome

DNA integration was found to take place randomly into the mitochondrial genomes of acute myeloid leukaemia samples, while specifically into the 5’- and 3’- UTRs of four proto-oncogenes, transcription of which is upregulated, supporting the researchers’ hypothesis that these integrations affect carcinogenesis of tumours within short distances of the microbiome.

They may however be "passenger mutations”, tumour cells may be simply more susceptible to LGT due to their rapid proliferation or potentially the bacteria are stimulating the transfer for their own benefit.

Interestingly, as illustrated in figures A and B (microbiome and LGT OTU read), above,

“In all of the cases examined, the composition of the microbiome across the samples is different from the composition of bacterial DNA integrated into the human genome.”

Helicobacter pylori infection is associated with a risk of stomach cancer, but in another beguiling inconsistency there was little evidence of this being its mechanism.

In closing the discussion, the authors write on the possibility that indeed the bacterial LGT is mutagenic,

“mutations that would benefit the bacteria would include those that create a micro-environment that promotes bacterial growth. This may explain why similar mutations, both in location and bacterial integrant, are observed in multiple individuals.”

Riley et al., "Bacteria-Human Somatic Cell Lateral Gene Transfer Is Enriched in Cancer Samples”. PLOS Computational Biology, vol 9, e1003107 (2013).

[rustbeltscientist]

Having not read the full paper, the mitochondrial results fit some of what we know about gene transfer.

1. In lower eukatyotes such as plants and fungi selfish genetic elements are exceedingly common for example about 50 % of fungi have a mitochondrial plasmid.

2. Gene transfer is also more common (at least in plants and fungi). RNA editing in plants is hypothesized to compensate for the rampant gene transfer that can occur (whole mtDNA in some plants). A recent finding in fungi has found a large region transferred into the mtDNA (Al-Reedy et al 2011). Mitochondria transfer between fungal cells and strains easily.

3. Animals apparently do everything to avoid these outcomes, removing "excess" mtDNA and reducing mitochondrial recombination for example. Mitochondria can be transferred between cells under limited circumstances however, they are also recognized as invaders when outside the cell triggering inflammation.

[deactivated-blog]

Father's Genetic Quest Pays Off

After a decade-long pursuit, one clinical geneticist has genetically diagnosed his own daughter’s rare conditions though his own efforts. By sequencing his daughter’s transcriptome along with the help of companies such as Illumina by exon-sequencing, he was able to shed more light on the “Rienhoff syndrome".

"Now nearly a decade into his quest, Rienhoff has arrived at an answer. Through the partial-genome sequencing of his entire family, he and a group of collaborators have found a mutation in the gene that encodes transforming growth factor-β3 (TGF-β3). Genes in the TGF-β pathway control embryogenesis, cell differentiation and cell death, and mutations in several related genes have been associated with Marfan syndrome and Loeys–Dietz syndrome, both of which have symptomatic overlap with Bea’s condition. The mutation, which has not been connected to any disease before, seems to be responsible for Bea’s clinical features, according to a paper to be published in the American Journal of Medical Genetics."

[rustbeltscientist]

Consensus sequences

I'm currently very interested in the idea of de novo sequencing and refining assemblies, as its something I've spent little time on. As far as getting a better consensus, I've seen manual realignment work well and poorly (Geneious). I've also read that both MAQ and picard tools will draw up a consensus, I'm interested to see how they compare. I have a theory that local realignment around indels can work but has anyone else tried it?