Next Gen Sequencing And RNAseq Flashcards
What are the NGS genomics methods?
Whole genome sequencing
Exome sequencing
Targeted sequencing
De novo sequencing
What are the NGS transcriptomics methods?
Total RNA sequencing
MRNA sequencing
Small NRA, noncoding RNA seq
What are the NGS epigenetic methods?
ChIP sequencing
Methylation sequencing
What are the steps in DNA library preparation?
Extract nucleic acids from blood, tissue, saliva etc
Shear dsDNA into fragments (300bp) and purify
Attach adapters to fragments
How do you sequence a DNA library?
Put DNA library on flowcell
-> bridge amplification
-> cluster amplification
Flowcell loaded onto a machine
What happens in sequencing-by-synthesis?
Annealing of sequencing primer
Controlled sequencing of each nucleotide 1 cycle at a time
Modified bases with reversible terminators and a different fluorescent dye tag
Single nucleotide incorporation
Flowcell wash
Imaging
Cleave chain terminator chemical group and dye with enzyme
Repeat for full length
What has to happen before library construction in RNAseq?
Conversion to cDNA
In RNAseq, what can be used as a measure of gene abundance?
Number of sequencing reads produced from each gene
What is the process of carrying out an RNAseq?
Experimental design
RNA prep
Library prep
Sequencing
Data analysis
What do the four lines in a standard fastq file show?
Sequence ID
Nucleotide sequence
Strand
Per base quality score
How do you analyse RNAseq data?
Align short sequence reads to the reference genome
Use specialist bioinformatic alignment programmes
What happens in the alignment file of an RNAseq?
Counting the number of mapped reads for sets of defined genomic intervals
What does a volcano plot show?
Genes that are up/down regulated in an RNAseq
Its a plot of log2 fold changes vs P-value for significance
What is gene set enrichment analysis?
Computational method that determines whether an a priori set of genes shows statistically significant, concordant differences between two biological states
