R Tutorial : Introduction to RNA-Seq

Key Takeaways

hi my name is Mary Piper I'm a consultant and trainer for the bioinformatics core at the Harvard th Chan School of Public Health my expertise is in RNA seek analyses and I'm excited to lead you through the RNA seek workflow in this course we will discuss all steps in the RNA seek workflow but we'll focus our hands-on lessons to the differential expression analysis by the end of this course you should be able to independently discover the genes that are differentially expressed between your experimental groups however before we get into the analysis details let's take a step back to ensure we have a solid understanding about what RNA seek is and the types of questions we can address using this technique all living organisms contain the instructions for life in their genome which is present in the nuclei of their cells the genome is comprised of double-stranded DNA divided into chromosomes for humans there are 23 but different organisms will have different different numbers of chromosomes the building blocks of our DNA are called nucleotides and there are four different nucleotide bases in DNA guanine adenine cytosine and thymine will refer to these nucleotides as G a C and T the double-stranded DNA forms a helix with a sugar phosphate backbone and within this helix a nucleotides pair with T and G nucleotides pair with C the order of these nucleotides is called the DNA sequence within this sequence are regions called genes genes provide instructions to make proteins which perform some function within the cell to make proteins the DNA is transcribed into messenger RNA or mRNA which is translated by the ribosome into protein some genes encode RNA that does not get translated into protein these RNAs are called non-coding RNAs or NC rnase often these RNAs have a function in and of themselves and include rrnas T RNAs and si RNAs among others all RNA is transcribed from genes are called transcripts to be translated into proteins mrna must undergo processing in this figure the top strand in the image represents a gene in the DNA comprised of the untranslated regions utrs highlighted in blue and the open reading frame highlighted in red genes are transcribed into pre mRNA which still contains the intronic sequences transcription represents the blue portion of this image after post-transcriptional processing shown in the gray section of this image the introns are spliced out in a poly a tail and five prime cap are added to yield mature mRNA transcripts the mature mRNA transcripts can be translated into protein shown in the red portion of the image while mRNA transcripts have a poly a tail which is a sequence of a's at the end of the transcript many of the non-coding rnas do not although all cells contain the same DNA sequence muscle cells are different from nerve cells and other types of cells because of the different genes that are turned on in these cells and the different RNAs and proteins produced similarly a disease-causing mutation can lead to differences in what genes are turned on or expressed and which genes are turned off a mutation can affect the type and quantity of RNAs proteins produced to explore the gene expression changes that occur in disease or between different conditions it can be useful to measure the quantity of RNA expressed by all genes using RNA seek then differential expression analysis of RNA seek data can be used to determine whether there are significant differences in gene expression between conditions using differential expression analysis we can ask various questions including which genes are different differentially expressed between sample groups are there any trends in gene expression over time or across conditions which groups of genes change similarly over time or across conditions what processes or pathways are enriched for my condition of interest now let's explore the biology of