Difference between revisions of "Eric"

Revision as of 14:55, 11 April 2017

January 19th Lab Methods in Genomics

Main Page

http://bio.davidson.edu/Courses/Bio343/LabMethods_2017.html

Story of Trisomy 21

Ts65DnDown Syndrome mouse model
T1DS/T2N -twin 1 down syndrome/twin 2 normal
MZ1/MZ2
-supernumerary
RPKM-reads per kilobase million -gives you a normal for comparison

LADs are overtranscribed in down syndrome when they should have low expression

Think replication time and transcription levels are correlated, genes transcribed earlier are expressed lower and later transcribed are more expressed

Not just the overexpression of chromosome 21. It is the consequence of these products causing the LADs to be expressed higher. Effects of changing chromatin structure

1/31

Molecular characterization of Ts65Dn

Ts65Dn is the mouse model for trisomy 21 in humans

distal= further away from the centromere

Think that chromosome 16 and 17 were combined via NHEJ but there is a small overlap region of 7 bp that might have had a role in determining the 16/17 breakpoint

writing as an introduction, not necessarily concerned with what we're going to do in the future

2/2 We are examining cDNA from the mouse models ~20 million reads per file

Separate each read based on barcode=3x nucleotide at end of chain

Trimmomatic used to delete barcode

use RSEM to map reads to genes and counts how many reads an identified gene is counted

DEseq2- enter name of data then click which data you want and run by execute

2/7

Use gene plot in galaxy after running DEseq 2. look for outliers and to search genes, use http://www.informatics.jax.org/batch/summary and delete decimals from gene number

disomic vs trisomic-looking for differences in expression, gene names, fold change=look at log2(FC), wald stats used to sort, base mean=reads per kilobase million

Which dataset is numerator?-first data set is numerator and second is denominator

Want to generate KEGG pathway maps for genes of interest, not looking at methylation differences, cant do with this data. We are looking at differential expression. Maternal Cont compared to Maternal Ts65Dn

to remove decimals in excel =left(A1, 18)

Save tab files as opening in excel and use excel to sort and whatnot. p-value cutoff=0.05 adjusted p-values are adjusted for the multiple tests that are performed

We will use the p-values with a value of less than 0.0001. This provides 105 genes of interest in the dataset of maternal trisomic vs maternal disomic

We took the genes and ran them through the naming program and export to excel

2/9/17 gene interaction tool

Protein map

Sorted genes of interest by chromosome location

log2(FC)= 2^that number = how much more expressed gene is

Filtered dataset by p-value of less than 0.0001 using workflow in galaxy, then sorted these data on log2(FC) greater than abs(.5)-using workflow to add filters and trims, etc.

2/16-working with visualizations in galaxy and after having found genes of interest, we are looking through these genes, trying to hone in on a few. We are using the p-values and fold change to determine which genes are expressed differently in the trisomic vs disomic

also, how do we get rid off the control variables between maternal and paternal

We are comparing Disomic maternal vs disomic paternal first to get a baseline difference in source dna and expression levels. Then we are comparing Trisomic maternal vs trisomic paternal to find differently expressed genes between these two. The next comparison will be between the Trisomic comp vs disomic comp to eliminate any differently expressed genes due to simply maternal vs paternal sources. setting our baseline from the disomic comparisons. We also will compare trisomic maternal to disomic maternal and trisomic paternal to disomic paternal

https://www.ncbi.nlm.nih.gov/pubmed/27243896

https://www.ncbi.nlm.nih.gov/pubmed/27538963

https://www.ncbi.nlm.nih.gov/pubmed/27029737

filter 1:c6<0.0001 filter 2: c3>=0.5 or c3<=-0.5 trim: column 1 everything to 18

Compared Trisomy maternal vs trisomy paternal, this resulted in 99 differently expressed genes, 10 of which were also differently expressed in the trisomy maternal vs disomic maternal. The whole dataset of trisomic paternal and maternal was run against the disomic maternal and paternal

could use two sample t test or one sample to explore stats of these differences between trisomic maternal and paternal and trisomic vs disomic

Genes that were found in all 3: Brwd1 Cct8 Cryz Dyrk1a Gabpa Jam2 Ltn1 Urb1

3/14/17- for individual status report: rewrite intro based on feedback, write what methods you've done, dont focus on discussion, discuss results you have. lots of visuals and data and be able to explain figures and data

Genes in depth

Dyrk1a-related to many down syndrome symptoms and phenotypes 

Alzheimer's
Using Epigallocatechin-3-gallate treatment to correct some craniofacial features EGCG
cancer resistance
target for chemotherapy
growth issues in DS
Dyrk1a effects on neurogenesis

CCT8

UCSC info

https://www.ncbi.nlm.nih.gov/pubmed/28342823 https://www.ncbi.nlm.nih.gov/pubmed/28096629 https://www.ncbi.nlm.nih.gov/pubmed/28069794 http://www.nature.com/nrg/journal/v18/n3/full/nrg.2016.154.html

TIAM1 -cardiac muscle hypertrophy, Wnt signalling pathway

Heart defects and tiam1
Congenetial heart failure and tiam1
contributes to heart defects in a gene dosage manner

greater methylation in general in DS, along with faster fetal acquisition of methylation-RUNX1, which is crucial for hematopoietic stem/progenitor cell, NKT and T-cell development.

Information about ESCs

Changing Epigenetics during differentiation-implies that this might not be the best model due to epigenetic changes that would occur in these cells down the line in development -gain of histone modification is tissue specific and associated with a loss in methylation, the opposite is rarely seen. hypomethylation at specific loci are important in cell lineage fate

Sex Specific Effects on ESC transcriptome and epigenome -hormonal influence on epigenetic regulation, can be carried on even with new neuronal genesis

These genes were differentially expressed between maternal and paternal trisomic *in ascending order (MTvsPT): Prmt9 Sumo2 Flot2 Aftph Gsn Dab2ip Chm Syncrip Rprd2 Wipf3 Stag1 Prmt5 Col3a1 Snrnp48 Wdr19 Zdhhc4 Ipo9 Zgpat Sh3tc1 Cib1 Arpp19 Cse1l Zfp207 Dolpp1 Slc38a3 Ccnd3 Gm10222 Pmvk Rab6b Sohlh2 Elavl4 Cep85 Serpinb6a Matr3 Prdm5 Gas7 Shroom4 Tom1l1 Nop56 Kank1 Mrps10 Baz2b Wnk1 Ubap2l Eloc Prrc1

mt-Atp6 Fubp1 Mcm10 Chm Srsf5 Ezh2 Cyb5b Zkscan5 Hmgn2 Mark2 Matr3 Zfp207 Vps51 Gstm1 Zfp12 Fubp3 Slit2 Ube2k Gatb Apbb2 Rangrf Acox1 Spata5 Senp6 Rps13 Zc3h11a Dohh Dnajc11 Actn4 Gcat Hnrnpdl Elavl4 Fgfr1 Mtx1 Bptf Ccdc136 Tbc1d24 Ptp4a3 Psmd2 Rcor3 Esrrb Kif20a Zfp993 Crocc D930048N14Rik Fus Ikbkg Relb Ing5 Asap1 Cryz

Why do we see so many genes differentially expressed between maternal trisomic and paternal trisomic that are not on the triplicated region of 17^16? What role do the triplicated genes play on the expression of these genes on other chromosomes. Does the triplicated region affect the geography of the nuclear DNA enough to cause these changes?