small. Since the samples were reliably normalized and belong to the same data set, the Value statistic is likely preferable. Describe a statistical test that would be appropriate for testing whether a significant difference exists between the levels of expression of the three genes that we are considering here between the two samples. In general, either a t-test family comparison (if a normality assumption is justified by the number and distribution of the data) or a Mann-Whitney U test (given more than ~5 samples per group) would be appropriate here. To simplify our analysis of the balance of fermentation to TCA cycle, we act on the log2 ratios of CS/LDHB levels in each sample. Observing that the log ratios are not normally distributed, we use the Mann Whitney U test, obtaining p=0.0007907 and a confidence interval of 0.29 0.97, thus suggesting a roughly 50% increase in – fermentation in the metastatic cells. Exercise 2 Analysis of high-throughput sequencing data – For the remainder of this lab session, you will work through a series of steps that could be applied to analyze a typical Illumina sequencing experiment. Typically, you would need access to a cluster or server with all of the described software installed on it. For simplicity, today we will simulate that situation be listing off the command line that would be used at each step, and having you act on only a small subset of the data to see what occurs at each step. Download the bundle containing the files that we are analyzing, and unzip it in a suitable location. The files are available on the course website. Quality control Given an initial set of fastq files from a sequencing run, you might run fastQC on those files as follows. fastqc reads.fastq The fastQC plot in fastqc_example.pdf shows a run where several QC metrics failed. Inspect the results, consulting the fastQC documentation at as needed. What to be the underlying cause for the QC problems? How would you recommend preprocessing the data before proceeding? The per-cycle base pair compositions indicate the presence of a single, strongly over-represented sequence. The sequences” heading shows that “Over-represented
small. Since the samples were reliably normalized and belong to the same data set, the Value statistic is likely preferable. Describe a statistical test that would be appropriate for testing whether a significant difference exists between the levels of expression of the three genes that we are considering here between the two samples. In general, either a t-test family comparison (if a normality assumption is justified by the number and distribution of the data) or a Mann-Whitney U test (given more than ~5 samples per group) would be appropriate here. To simplify our analysis of the balance of fermentation to TCA cycle, we act on the log2 ratios of CS/LDHB levels in each sample. Observing that the log ratios are not normally distributed, we use the Mann Whitney U test, obtaining p=0.0007907 and a confidence interval of 0.29 – 0.97, thus suggesting a roughly 50% increase in fermentation in the metastatic cells. Exercise 2 – Analysis of high-throughput sequencing data For the remainder of this lab session, you will work through a series of steps that could be applied to analyze a typical Illumina sequencing experiment. Typically, you would need access to a cluster or server with all of the described software installed on it. For simplicity, today we will simulate that situation be listing off the command line that would be used at each step, and having you act on only a small subset of the data to see what occurs at each step. Download the bundle containing the files that we are analyzing, and unzip it in a suitable location. The files are available on the course website. Quality control Given an initial set of fastq files from a sequencing run, you might run fastQC on those files as follows. fastqc reads.fastq The fastQC plot in fastqc_example.pdf shows a run where several QC metrics failed. Inspect the results, consulting the fastQC documentation at as needed. What to be the underlying cause for the QC problems? How would you recommend preprocessing the data before proceeding? The per-cycle base pair compositions indicate the presence of a single, strongly over-represented sequence. The “Over-represented sequences” heading shows that
module3_lab2_solutions.docx