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Revision as of 15:22, 14 February 2017

January 19th Lab Methods in Genomics

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.