Computational Skills Courseweek 4

Mike GilchristNIMR May-July 2011

WEEK FOUR

Individual project plans

Laurent

RNA seq -Reads from solexa

Alignment on the transcriptome

Normalization

Analysis of the transcriptional dynamic of chosen gene regulatory networks

P tc h1

0

1000

2000

3000

4000

9.5 10 10.5 11

forelimb

hindlimb

S hh

0100200300400500

9.5 10 10.5 11

forelimb

hindlimb

Aims of the project :

- Comparing the transcriptional profiles of forelimbs and hindlimbs over an embryonic time-course:

- Identify additional candidates “limb-type modifiers”

- Compare the transcriptional profile dynamics between FL and HL of common forelimb/hindlimb GRNs to establish a limb-type signature

Veronique

From about E11.5:reduced Lhx6 levels

After P60:Spontaneous

Seizures

From E13.5:reduced Sst levels P40 - P60:

Physiological defects at inhibitory dendritic synapses

Our lab generated an hypomorphic Lhx6 allele which expresses reduced levels of mRNA. This allele specifically affects differentiation of a subset of cortical interneurons. This results in the development of seizures. These unique mutants allow the study of mechanisms of specification of cortical interneuron subtypes and the generation of seizures.

P15: mRNA-Seq experimentmRNA extracted from cortex+/- : controls (4)-/-: nulls (3)LacZ/-: hypomorphs (3)Questions:1) Molecular processes affected2) Molecular markers forcell types affected

Guilherme

How do binding sites for the T-box transcription factor Brachyury change over time during frog embryogenesis?

George

Data• mRNA-seq in chick neural cells + and - Shh• database of chick transcription factors (TFs)• ChIP-seq analysis identifying binding sites of several TFs in

mouse neural cells responding to Shh

Analysis• map mRNA-seq data to chick genome• measure gene expression levels and identify differentially

expressed genes• identify subset of regulated genes that are TFs

• identify mouse orthologs of differentially expressed TFs (and genes)

• identify clusters of TFs binding near regulated genes• ask whether there is (i) any enrichment for clusters of binding

sites near regulated genes; (ii) any correlation between combination of Tfs bound and type of regulation; (iii) predictive value in the ChIP-seq data for the regulation of gene expression.

James

The ‘PROJECT’ - Siggi Sato (Parasitology)

Gene expression in P. falciparum

Nuclear genome

Identify genetic elements determining the limit of the

intron

Protein/RNA factors for splicing and controlling organelles

Organellar genomes (Plastid, Mitochondrion)

Identify genetic elements for replication and transcription

New anti-P. falciparum

Finding new substances and identifying their targets

Alaremycin (patent filed)

MRC-T “small molecules”

( Others? )

Siggi

http://www.ensembl.org/Mus_musculus/Location/

The molecular regulation of IL-10 and IL-12 in innate cells: Investigating the differential The molecular regulation of IL-10 and IL-12 in innate cells: Investigating the differential production of IL-10 and IL-12 in commonly used inbred mouse strainsproduction of IL-10 and IL-12 in commonly used inbred mouse strains

• Key initial questions include:

o Are there differences (SNPs/deletions) in the IL-10/IL-12/type I IFN loci?o Are these differences in regulatory elements (TF binding sites/3’UTR) or protein coding regions?

Ashleigh

Are there any genes in the Xenopus tropicalis genome

that do not have a corresponding EST in the Xenopus laevis database?

& Vice versa

Alex

DB IMGT RF http://imgt.cines.fr/cgi-bin/IMGTlect.jv?query=5+AB019437 AC AB019437 SP Human GL IGHV GN V7-81 NA caggtgcagctggtgcagtctggccatgaggtgaagcagcctggggcctcagtgaaggtc NA tcctgcaaggcttctggttacagtttcaccacctatggtatgaattgggtgccacaggcc NA cctggacaagggcttgagtggatgggatggttcaacacctacactgggaacccaacatat NA gcccagggcttcacaggacggtttgtcttctccatggacacctctgccagcacagcatac NA ctgcagatcagcagcctaaaggctgaggacatggccatgtattactgtgcgagata AA QVQLVQSGHEVKQPGASVKVSCKASGYSFTTYGMNWVPQAPGQGLEWMGWFNTYTGNPTY AA AQGFTGRFVFSMDTSASTAYLQISSLKAEDMAMYYCAR//

Flat file database of mouse and human sequences from databases IMGT, ABG, NCBI and VBASE2 in EMBL format.

Load (how?) into MySQL (database design: tables and primary key?)

Remove redundancy in sequences but retain pointers to other fields.

Flexible query and output different sets of sequences for e.g. blast search.

Jose

Identify differences in histone patterns between IL-10

secreting vs non-secreting T helper cells

FACS purifyCD4+CD44loCD25-Foxp3GFP-10BiT-

T cells from SPN

Culture with: - HEL peptide - DCs - Skewing cytokines/ blocking antibodies

FACS purifyCD4+10BiT+

vsCD4+10BiT-

T cells10BiT (IL-10 reporter) Foxp3GFPTCR7 Rag1-/-

ChIP-Seq: Histone Modification

IL-10 Associated Histone Modification Pattern of T helper Subsets

Gene status Histonepattern

Permissive TSS H3K4me3

Transcribed gene H3K4me3 + H3K36me3

Bivalent domain H3K4me3 + H3K27me3

Repressed TSS H3K27me3

Poised enhancer H3K4me1

Active enhancer H3K4me1 + H327Ac

Histone Modification Pattern Maps

List of histone marks of all genes in the different subsets

Assign histone patterns to genes in the different T helper cell subsets

Compare histone patterns in the different T helper cell subsets;

gaining insight into “housekeeping” vs activation vs lineage defining

genes

Foxp3

Rort

Tbet

Gata3

IL-10?10BiT+ 10BiT-

IL-

4,

IL-6

, IL

-12,

IF

N-

, TG

F

IL-4, IFN-,

IL-12

IL-12, IL-4

TGF, IL-6,IFN-, IL-4

TGF, IL-2

IL-4IL-10

IFN-IL-10

TGFIL-10

IL-17IL-10

IL-2

Leona

Khokha Lab – Computational Goal• Define All Exons in X. tropicalis• How?

• Combine current gene models, transcriptome assemblies, available and soon to be available RNA-seq

• Why?• Genome sequence/annotation - imperfect• Exon Capture Sequencing – mutant gene

identification• Gap Capture – gene model improvements• Analysis of RNA-seq data

• When?• Tomorrow would be good

Mustafa

Mary

One exercise I am trying now is to predict the potential PfSUB2 (a subtilisin-like protease with relative sequence-specificity at the cleavage site) in Plasmodium falciparum protein database. I downloaded predicted  protein sequences for one chromosome (Chr 13) and  by using grep detected 38 sequences. After editing output with sed to make it look like a fasta file (line numbers as identifiers for each sequence), queried against the chr13 protein database (blastp for max_target_seqs 1) to obtain accession numbers.

I am yet to write/try any script.

What I would like to do: I have a Pfmsp7 knock out parasite line which shows invasion phenotype. Once the technical hurdles are passed (like getting rid off rRNA sequences with polyA in it), we want to RNAseq analyse in order to identify genes that may have been affected.

Madhu