Functional annotation of farm animal genomes: ChIP-seq.
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1 Functional annotation of farm animal genomes: ChIP-seq Richard Crooijmans 2018, PAGXXVI
2 Why FAANG is important Understanding the genotype to phenotype link This needs: - genomic selection - improving fundamental understanding of biology - high quality reference genome sequence - comprehensive annotation of the functional elements - common infrastructure providing - biological resources - Bioinformatics tools - databases
3 What to study?
4 International reference material Pig IPEC-J2 Established from normal intestinal epithelium cells isolated from the jejunum of a neonatal, unsuckled pig Available from DSMZ in Germany Chicken SL-29 Established from a 11 day old decapitated chicken embryo by standard trypsinization Layer (leghorn) Available from ATCC Both primary untransformed cell lines
5 FAANG assays Whole genome re-sequence (WGS: 30x) RNA-seq, poly A and stranded Whole genome bisulfite sequencing (WGBS: 50x) Reduced representation bisulfite sequencing (RRBS) ChIP-seq (histone marks and CTCF) H3K27ac H3K4me3 H3K27me3 H3K4me1 CTCF active enhancers and promoters promoter active genes and transcription start sites silenced genes (active during developmental stages) active enhancers insulator activity ATAC-seq (University of Leiden, The Netherlands; in progress using different protocols)
6 Preliminary analysis Standard analysis WGS: BWA mem RRBS: BSeeker2 WGBS: BSseeker2 RNA-seq: Tophat and Cufflinks ChIP-seq: Bowtie and MACS Future: FAANG analysis pipelines
7 Methylation Whole Genome Bisulphite Sequencing (WGBS) High coverage needed Quiet expensive Reduced Representation Bisulphite Sequencing (RRBS) Much lower cost Enrichment of the specific regions
8 Methylation: Comparison WGBS and RRBS Comparison of overlapping sites (Coverage >10 reads) Similar methylation distribution Very good correlation between 2 RRBS samples (0.96) Good correlation between WGBS and RRBS (0.93) WGBS RRBS WGBS RRBS
9 Methylation: RRBS and WGBS provide complementary information WGBS RRBS WGBS RRBS HOXA4 Region with very low coverage in WGBS but sufficient coverage in RRBS data Chr 18:50,074,454-50,078,736
10 ChIP-seq Active elements that interfere in gene expression (enhancers, silencers) Different protocols Extracting nuclei Fixation Amount of input for ChIP experiment Antibodies Different controls in the procedure (positive and negative) 10
11 Example expressed gene: SCO1 Signal for H3K4me3, H3K27ac and H3K4me1 (active enhancer and promoters) No signal for silent neighboring gene H3K27me3 bcall H3K4me3 ncall H3K27ac bcall H3K4me1 bcall Gene expression H3K4me3 cov. H3K27ac cov. H3K4me1 cov. H3K27me3 cov. SCO1 ADPRM chr 12:58,037,835-58,069,194
12 Example silenced genes (HOX genes chr18) Many H3K27me3 peaks: Silenced genes H3K27me3 bcall H3K4me3 ncall H3K27ac bcall H3K4me1 ncall Gene expression H3K4me3 cov. H3K27ac cov. H3K27me3 cov. H3K4me1 cov. HOXA6 HOXA5 HOXA4 A promoter element but not active due to missing H3K27ac? 18:50,054,955-50,084,206
13 Example CTCF consensus at CTCF peak
14 qpcr: Positive controls for H3K4me3 and H3K27ac NDUFA4 expression H3K4me3 H3K27ac NDUFA4 gene, minus strand primers TRX1 expression H3K4me3 H3K27ac TRX1 gene, minus strand primers
15 qpcr: Negative control primers (gene desert) No signal for any of the histone and CTCF marks 0-10 No Gene expression primers Chip_NegC2_2 position 2:44,492,350-44,498,036
16 qpcr: Positive control for H3K27me3 at HOX genes Primer combinations not very good: design new combinations Gene expression H3K27me3 primers HOXC4 Gene expression H3K27me3 HOXB5 primers
17 Next step: from cultured cells to tissue * Optimize protocols extracting nuclei - from different tissue - from different cells - from limited number of cells/tissue (biopsies, organoids) * Optimize controls qpcr - positive and negative controls per tissue * Reports and protocols on the FAANG website * Samples deposited in Biosamples (EBI)
18 Overview ChIP seq assays Species Tissue/cell line name Mark Ab provider reference Ab quantity (µg) starting amount/ip chromatin preparation Frozen Tissue Frozen Tissue Frozen Tissue Frozen Tissue Frozen Tissue H3K4me3 Diagenode C ,5 and 1 ug 2mg of tissue DIAGENODE_SOP_ChIPseq_for_Histone_Marks_ H3K27ac Diagenode C ,5 and 1 ug 2mg of tissue H3K27me3 Diagenode C ,5 and 1 ug 2mg of tissue DIAGENODE_SOP_ChIPseq_for_Histone_Marks_ DIAGENODE_SOP_ChIPseq_for_Histone_Marks_ H3K4me1 Diagenode C ,5 and 1 ug 2mg of tissue DIAGENODE_SOP_ChIPseq_for_Histone_Marks_ H3K4me3 Diagenode C ,5 and 1 ug 50mg of tissue DIAGENODE_SOP_ChIP-seq_for_low_input_ Frozen Tissue H3K27me3 Diagenode C ,5 and 1 ug 50mg of tissue DIAGENODE_SOP_ChIP-seq_for_low_input_ Frozen Tissue H3K27ac Diagenode C ,5 and 1 ug 50mg of tissue DIAGENODE_SOP_ChIP-seq_for_low_input_ Frozen Tissue H3K4me1 Diagenode C ,5 and 1 ug 50mg of tissue DIAGENODE_SOP_ChIP-seq_for_low_input_ Chicken Cell H3K4me3 Diagenode C μg 5x 10 6 cells WUR_SOP_ChIP-seq_ Chicken Cell H3K27me3 Diagenode C μg 5x 10 6 cells WUR_SOP_ChIP-seq_ Chicken Cell H3K27ac Abcam ab μg 5x 10 6 cells WUR_SOP_ChIP-seq_ Chicken Cell H3K4me1 Abcam ab μg 5x 10 6 cells WUR_SOP_ChIP-seq_ Chicken Cell CTCF Merck μl 5x 10 6 cells WUR_SOP_ChIP-seq_ Pig Cell H3K4me3 Diagenode C ug 5x 10 6 cells WUR_SOP_ChIP-seq_ Pig Cell H3K27me3 Diagenode C ug 5x 10 6 cells WUR_SOP_ChIP-seq_ Pig Cell H3K27ac Abcam ab μg 5x 10 6 cells WUR_SOP_ChIP-seq_ Pig Cell H3K4me1 Abcam ab μg 5x 10 6 cells WUR_SOP_ChIP-seq_ Pig Cell CTCF Merck μl 5x 10 6 cells WUR_SOP_ChIP-seq_
19 ChIP-seq: qpcr controls positive controls animal id development stage/ tissue/cell ChIP-seq primer _R primer_f primer _R Species (BioSamples) sex age s antibody Gene id genome build genome location primer_f primer_f annotation primer_r annotation Gene id primer_f annotation primer_r annotation Gallus gallus male cells all Ggallus5.0 2: :44,496,315 Negative control Sus scrofa male cells H3k27ac NDUFA4 Sscrofa : 30,448,569-30,449,050 Sus 13: 30,448,569- scrofa male cells H3K4me NDUFA4 Sscrofa ,449,050 Sus 1:251,264,178- scrofa male cells H3k27ac TRX1 Sscrofa ,277,550 Sus 1:251,264,178- scrofa male cells H3K4me TRX1 Sscrofa ,277,550 13:30,448,759-13:30,448,779 13:30,448,759-13:30,448,779 13: 30,448,838-13:448,848 13: 30,448,838-13:448,848 positive controls ChIP-seq antibody Gene id genome build genome location primer_f primer_f annotation primer_r primer _R annotation all Ggallus5.0 H3k27ac NDUFA4 Sscrofa11.1 H3K4me NDUFA4 Sscrofa11.1 H3k27ac TRX1 Sscrofa11.1 H3K4me TRX1 Sscrofa : 30,448,569-30,449,050 13: 30,448,569-30,449,050 1:251,264, ,277,550 1:251,264, ,277,550 ACGTGGATCG 13:30,448,759- CCTGAGTC 13:30,448,779 ACGTGGATCG CCTGAGTC 13:30,448,759-13:30,448,779 CCTGACGTCT 13: 30,448,838- GACCGTGA 13:448,848 CCTGACGTCT GACCGTGA 13: 30,448,838-13:448,848 19
20 Questions to the FAANG community 1. ChIP-seq protocols (fresh/frozen; cells/tissue/biopsy/single cell) a. tissue disaggregation and DNA-protein cross-linking b. Including tips and tricks /tissue/stage c. qpcr positive and negative controls/tissue/species Mail to: 20
21 Summer school Wageningen, June 25-29, 2018 Content: (pre-announcement) - International speakers on functional annotation. - Wet-lab experiments (hands-on) - Data analysis (data made available) 21
22 Thanks Martien Groenen Ole Madsen Geoffrey Berguet 22
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