If you are an Instagram user, at some point, you care going to be interested in the various metrics such as followers, number of posts by a certain user etc. You might want to compare these metrics between different users or to find out the number of posts with a certain hashtag etc. The casual way to do it is to go the relevant Instagram page and look at the metric and write it down somewhere, and go to next and so on. Clearly this is not ideal strategy if you want to look at a few hundred pages. It would be neat to get this data in an automated manner.
I use ImageJ for many of my image analysis needs. My desktop computer runs Windows 7 and it has pretty solid specs with Core i7 processor and 16GB RAM. I recently had to handle some large tiff stacks (4-5gb) and it simply wouldn’t work on my desktop as I constantly ran into ‘out of memory’ errors. So I decided to run them on a computing cluster instead since I have access to one. Running on a cluster might be useful when handling data with large memory requirements or to perform computations on numerous files in parallel by distributing load to multiple cores. It took me a while to figure out how to get things to work, so I thought I would make a record of it. And this might hopefully be useful to others.
In a standard statistical test, one assumes a null hypothesis, performs a statistical test and computes a p-value. The estimated p-value is compared to a predetermined threshold (usually 0.05). If the estimated p-value is greater than 0.05 (say 0.2), it means that there is a 20% chance of obtaining the current result if the null hypothesis is true. Since we decided our threshold as 5%, the 20% is too high to reject the null hypothesis and we accept the null hypothesis. Now, if the estimated p-value was less than 0.05 (say 0.02), there is a 2% probability of obtaining the observed result if the null hypothesis is true. Since 2% is a very low probability and it is below our threshold of 5%, we reject the null hypothesis and accept an alternative hypothesis.
The 5% threshold, although giving us high confidence, is an arbitrary value and does not absolutely guarantee an outcome. There is still the possibility that we are wrong 5% of the time. This is known as the probability of a Type I error. A Type I error occurs when a researcher falsely concludes that an observed difference is real, when in fact, there is no difference.
That was the story of a single statistical test. With large data, it is common for data analysts to do multiple statistical tests on the same data. Similar to a single test, each test in a multiple test has the 5% Type 1 error rate. And this accumulates for the number of tests.
It may seem like the world is descending into total chaos, violence, and destruction. War in Syria, Ukraine, Yemen, Islamic state, migrant crisis, Ebola, plane crashes, earthquakes, tsunamis and what-not. The more news you watch, the more worried you will be. This is because the news outlets tend to focus on spectacularly negative instances. Violence, atrocities, and hatred are thrown into the spotlight and into the lives of common people. With the ever increasing digital connectivity, it is easy to disseminate information and to absorb information at an unprecedented level. Relatively smaller incidents have a larger voice. As said by Ray Kurzwil, “The world isn’t getting worse, our information is getting better”. To appreciate the world we live in, we have to put things into a wider context.
The fact is that humanity has never lived in a better time than now in pretty much every aspect you look at; war, violence, diseases, poverty are all at the lowest it has ever been. Of course, there is still a long way to go, but this is the best it has been since the beginning of humankind. To prove my point, here we evaluate human progress using some real data and simple time-series plots. Most of the data and information was obtained from OurWorldInData.
In RNA-Seq analyses, adding pre-determined quantity of synthetic RNA sequences (spike-ins) to samples is a popular way to verify the experimental pipeline, determine quantification accuracy and for normalisation of differential expression. The most commonly used spike-ins are the ERCC spike-ins.
This post will cover the bioinformatic steps involved in obtaining read counts of spike-ins from a FASTQ file sequenced with spike-ins. The steps are namely creating a custom FASTA genome build incorporating the spike-in sequences, custom GTF file creation, mapping the reads to the custom genome, read counting and visualisation. This post will not be covering the wet lab part of adding spike-ins. I have a FASTQ data file (sample01.fq.gz) from single cell 50bp single-end Illumina reads with spike-ins that I am using for this workflow.