Category: Research Blog Page 4 of 7

Highly multiplexed and strand-specific single-cell RNA 5′ end sequencing

Tony Gamble and David Zarkower have a nice primer on sex determination in Current Biology.

As you all know, getting good in situs is a pain in the butt.  One problem is the difficulty in finding the optimal probe.  Here is a paper from Bill McGinnis’ lab that looks at some of the issues affecting probe quality, and a program that helps you pick the best in situ probes.

Abstract:

Fluorescent in situ hybridization (FISH) techniques are becoming extremely sensitive, to the point where individual RNA or DNA molecules can be detected with small probes. At this level of sensitivity, the elimination of ‘off-target’ hybridization is of crucial importance, but typical probes used for RNA and DNA FISH contain sequences repeated elsewhere in the genome. We find that very short (e.g. 20 nt) perfect repeated sequences within much longer probes (e.g. 350-1500 nt) can produce significant off-target signals. The extent of noise is surprising given the long length of the probes and the short length of non-specific regions. When we removed the small regions of repeated sequence from either short or long probes, we find that the signal-to-noise ratio is increased by orders of magnitude, putting us in a regime where fluorescent signals can be considered to be a quantitative measure of target transcript numbers. As the majority of genes in complex organisms contain repeated k-mers, we provide genome-wide annotations of k-mer-uniqueness at http://cbio.mskcc.org/ approximately aarvey/repeatmap.

Here’s a recent paper in PNAS that looks at the use of viral 5′ and 3′ UTRs to enhance gene expression from UAS trangenes.  They show that using particular viral and other heterospecific UTRs can seriously increase protein abundance, and thus give stronger phenotypes.

These tricks are worth considering if you are making UAS or Lex constructs for ectopic geen expression.  More immediately, the enhanced UAS-shi[ts] construct they made can be useful for some of your neuronal silencing experiments.

Quote: “In this report, we demonstrate that sequences derived from the 5′-UTR and 3′-UTR of viral mRNAs, as well as from the abundantly expressed lobster tropomyosin gene (35), are able to function in Drosophila to enhance protein production. By using 5′- and 3′-UTR elements in combination, increases of >20-fold have been achieved, allowing single transgenes to achieve protein expression levels that previously required multiple transgenes, thereby greatly facilitating genetic strain construction. We also show that the 3′-UTR from the Autographa californica nuclear polyhedrosis virus (AcNPV) p10 gene functions efficiently in the female germline. “

There’s a paper from Hopi Hoekstra’s lab coming out in Evolution, where they look at the genetics of pigmentation in their favorite oldfield/beach mouse populations. The main point of the paper is a comparison between population history estimated from multi-locus data and the phylogeny of the causative gene that causes color variation.  In addition, take a look at the methods – see how they used a combination of sequence capture and barcoding to sequence several thousand random loci as well as a very large region surrounding Mc1R.

A recent paper in Nature Biotechnology has a performance comparison of benchtop high-throughput sequencing platforms.  These “small-scale” (hundreds of megabases to a few gigabases) machines will probably be increasingly prevalent, and useful for our kind of work, so check it out.

And speaking of John Yoder: here is his video protocol in JoVE for abdominal dissections and immunohistochemistry.  This should be useful to some of you.

John Yoder has just published a new paper looking at the interaction between dsx and Abd-B in sculpting the posterior abdominal segments in males vs females.  The posterior abdomen is clearly sexually dimorphic, and in some respects the roles of dsxM and Abd-B in the abdomen are similar to the roles of dsxM and Scr in the sex comb.  In particular, dsx expression is regulated by Abd-B.   But there are also important differences – for example, Abd-B does not seem to be affected by Dsx.  Check it out.

The latest issue of NRG has a primer on genome annotation principles and software:

http://www.nature.com/nrg/journal/v13/n5/full/nrg3174.html?WT.ec_id=NRG-201205

Here is another attempt at the melanogaster group phylogeny:

http://www.ncbi.nlm.nih.gov/pubmed/21985965

Unfortunately, it does not tell us anything we don’t already know.  Let us hope that a proper phylogenomic analysis using the new genome sequences will finally nail down those pesky internal nodes.