Genomics, Transcriptomics, and Development

YASS

Limitations of techniques used to study genes and proteins

These approaches typically have been restricted to studying a limited number of targets

Twin Studies

Quantifies phenotypic differences in monozygotic (genetically identical) and dizygotic (genetic similarity) twin pairs to calculate concordance and estimate heritability. Nature vs nurture?

Twin Studies Concordance for Autism MZ pairs (n==11)

36%

Twin Studies Concordance for Cognitive Disorder (including autism) MZ pairs (n==11)

82%

Heritability (h²)

a measure ranging in value from 0 to 1

H = 1

all variation is due to differences in genotype

H = 0

all variation due to differences in the environment

Twins study H²

Values range from .41 - .79 suggesting both environment and genotype can influence

Sanger sequencing

• chain-terminating dideoxynucleotide triphosphates (ddNTPs) that stop DNA polymerization during PCR to cleverly resolve sequence by measuring size

• need regular dNTPs + fluorescently-conjugated ddNTPs and Taq polymerase

• PCR initiates generation of fragments then fluorescent ddNTP is incorporated, amplification of the strand stops and is color coded.

• size separation by gel electrophoresis; up to 1000 bp can be determined

Whole Genome Sequencing

the comprehensive sequencing of the entire genome

Human Genome Project

completed the first WGS of ~3 billion bp

What is transcriptomics and its evolved techniques?

studying expression of all genes

• microarrays

• next-generation sequencing

• single-cell RNA-seq

• spatial transcriptomics

Microarrays

attach small oligonucleotides to multiple genes of interest on a chip; by 2000s microarrays containing ~20,000 oligonucleotides complementary to all known protein-coding mRNAs were regularly used to study the transcriptome

• Each dot = a collection of oligonucleotides complementary to a specific gene's mRNA

Single Nucleotide Polymorphism (SNP)

a genomic variant at a single base position in the DNA; used to conduct Genome Wide Association Studies (GWAS) that detect associations between genetic loci and traits.

GWAS

90-95% of SNPs detected in GWAS are located in non-coding genomic regions that indirectly modulate gene expression ~40-50% of the time.

Next Generation Sequencing Technologies Approaches

DNAseq & RNAseq

Massively parallel sequencing allows for simultaneous processing of multiple DNA or RNA fragments.

RNAseq

1. Fragment RNA into smaller pieces.

2. Convert fragments to cDNA using reverse transcription and random priming.

3. Ligate adaptors to the cDNA.

4. Amplify the cDNA.

5. Sequence the prepared library.

NGS Example: Reversible Dye-Terminator (RDT)- based sequencing-by-synthesis (SBS)

1. fragments of sample DNA/RNA template of interest are anchored to chip;

2. RDT-ddNTPS fluorescently labeled are added during synthesis and picture is taken at end of each cycle

3. enzyme cleaves tag and turns RDT-ddNTP into dNTP to reverse termination and repeat (100-1000 bp each)

Bioinformatics and Resequencing

~100 GB .fastq file containing ~3 billion bases in the human genome

Resequencing: when reads are aligned back to known genome sequence to identify all genes/ variations, including: SNPS, insertions/deletions, structural variants, copy number variants)

Public Repositories and RPKM

provide RNAseq based information on gene expression

Reads Per Kilobase transcript per Million mapped reads

Single Cell RNAseq (scRNAseq)

By gently digesting/dissociating tissue, single cells/nuclei can be extracted and nucleic acids from individual cells/nuclei can be sequenced

Spatial Transcriptomics

Isolation of single cells in scRNA-seq destroys information on spatial localization and proximities to other cells.