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What is the quality of my RNA-seq reads? Are my RNA-seq reads of good quality or are there artifacts and errors I should remove and/or be aware of?

This recipe provides an outline of one method to preprocess and determine the quality of RNA-seq libraries. An example use of this recipe is a case where an investigator has completed sequencing of samples, and wants to evaluate and check the quality of the sequencing results before going through the time-consuming process of aligning the reads against a genome.

 

Given a set of raw RNA-seq reads, the goal is to align the reads to a reference genome and assess the quality of the read alignments by obtaining metrics such as depth of coverage, rRNA contamination, continuity of coverage, and GC bias. The purpose of this recipe is to process raw RNA-seq reads prior to aligning them against a reference genome. In particular, this recipe uses a dataset which has RNA-seq reads contaminated with adapter sequences. First we identify and remove adapter sequences using Galaxy, then we process the data and align it to a reference genome using GenePattern.

Why remove adapter sequences? Adapter sequences are short pieces of DNA with known sequence, which are used to link DNA molecules together. In particular, these are used to link an unknown DNA sequence to a sequencing primer, in order to sequence the unknown DNA strand. Most of the time adapter sequences are removed during pre-processing when data is sent to a researcher; however, sometimes some adapter sequences remain. It is important to remove these sequences before aligning reads to a genome.

 

Which genes are differentially expressed between my two phenotypes, based on my RNA-seq data?

This recipe provides one method to identify differentially expressed genes in RNA-seq read data. An example use of this recipe is a case where an investigator may want to compare two phenotypes, such as two types of cancer, to determine which genes are up- or down-regulated differently between these phenotypes.

 

In particular, this recipe uses the UCSC Table Browser to retrieve a reference genome to align RNA-seq reads against. We also used several modules in GenePattern to align the reads against the reference genome, and to identify differentially expressed genes when comparing two conditions. Finally, we use IGV to visualize the differentially expressed genes.

Why differential expression analysis? We assume that most genes are not expressed all the time, but rather are expressed in specific tissues, stages of development, or under certain conditions. Genes which are expressed in one condition, such as cancerous tissue, are said to be differentially expressed when compared to normal conditions. To identify which genes change in response to specific conditions (e.g. cancer), we must filter or process the dataset to remove genes which are not informative.

 

 

Which genes are highly expressed, based on my RNA-seq data? Are any of the highly expressed genes also differentially expressed?

This recipe provides an outline of one method to identify and visualize genes and isoforms that are highly expressed in RNA-seq data. In particular, this recipe utilizes an analysis pipeline, allowing a user to chain together multiple analysis steps into one workflow that can be run in one step. An example use of this recipe is a case where an investigator wants to process several datasets in the same way, in which case the pipeline will allow the investigator to re-use the same modules and parameters, over and over again.

 

Given a set of raw RNA-seq reads, the goal is to align the reads to a reference genome, estimate expression abundance levels for reference genes and isoforms, filter out low-expressed genes and isoforms, and visualize the read alignments and their expression levels. In particular, this recipe uses the UCSC Table Browser to retrieve a reference genome to align RNA-seq reads against. We also uses several modules in GenePattern to align the reads against the reference genome, and to identify differentially expressed genes when comparing two conditions. Finally, we use IGV to visualize the differentially expressed genes.

Why differential expression analysis? We assume that most genes are not expressed all the time, but rather are expressed in specific tissues, stages of development, or under certain conditions. Genes which are expressed in one condition, such as cancerous tissue, are said to be differentially expressed when compared to normal conditions. To identify which genes change in response to specific conditions (e.g. cancer), we must filter or process the dataset to remove genes which are not informative.

 

Does my RNA-Seq data contain noticeable batch effects? Can I remove any batch effects I find?

This recipe provides one method to assess RNA-seq count data for potential batch effects. Identified batch effects are removed using the ComBat algorithm (Johnson, Rabinovic, and Li). An example use case of this recipe is when an investigator wants to correct data and account for confounding variables generated by effects such as using different reagents or processing samples at different times.

 

This recipe uses count data and sample annotations derived from two separate RNA-seq experiments and relies upon Principal Component Analysis (PCA) as a primary means of evaluating the dataset for batch effects. In particular, this recipe utilizes a number of GenePattern modules to filter and preprocess the RNA-seq count data and to normalize the dataset using the ComBat algorithm. We then use Galaxy to view the influence of batch effects in our pre- and post-normalization datasets.

Why consider batch effects (a.k.a. confounders)? A "batch" or "confounding" variable can be generally defined as an extraneous variable that may correlate or anti-correlate with our variables of interest. Failing to account for confounding variables or batch effects can lead us to draw spurious conclusions from our data. For example:

Albert is comparing the hardiness of two different E. coli strains, X and Y, by measuring their production of various stress proteins under a mild bactericide. However, Albert's bactericide stock is running low and he ends up using two different stocks of the same bactericide over the course of this experiment. Here, bactericide stock is a confounding or batch variable. Perhaps one bactericide stock is more potent than the other. Albert will need to be careful in how he analyzes his data. He must somehow ensure that differences in stress protein abundances between his X and Y strains are driven by the biological differences of these two strains rather than differences in the bactericide stocks.

Efforts can be undertaken to minimize batch effects by ensuring proper experimental design, e.g., simplifying protocols to eliminate potential confounders. If batches are unavoidable, it is also important to randomize samples across these batches such that differences between batches can be attributed to batch effects rather than biological differences. But despite these efforts, unforeseen batch effects may still be present in any experiment. Thus, we should be aware of the degree to which these unwanted factors influence our results and be prepared to use various statistical methods to remove these unwanted factors if needed.

How do we identify batch effects? There are a variety of methods for identifying batch effects. Sometimes, potential batches can be identified at the outset of a project based on its experimental design. For example, if samples must be processed in separate groups due to the limiting capacity of a sequencer, then these processing groups are likely to introduce batch effects. If batches can be pre-identified, then the scientist can include identical control samples in each batch. Any variation in measurements from these control samples can then be attributed to and used to quantify a batch effect.

Another common method is to visualize data using a PCA projection. PCA uses a linear transformation to transform a dataset into a space of linearly uncorrelated, orthogonal "principal components". More simply, PCA is a way of visualizing data such that underlying structures are revealed. We encourage users to learn more about PCA (Ma and Dai's 2011 article in Briefings in Bioinformatics is a good starting point). By re-projecting with PCA, we can identify those variables, including batch variables, that are contributing to large variances in the dataset.

How do I obtain and analyze data from The Cancer Genome Atlas (TCGA)? Which TCGA datasets have specific mutations in my gene of interest?

This recipe provides a method for identifying and obtaining specific datasets of interest from The Cancer Genome Atlas (TCGA), through a web-based tool called FireBrowse. An example use of this recipe is a case where an investigator may have a gene they are interested in, such as ERCC2, and would like to know if there are mutations in this gene in specific datasets of interest, such as bladder cancer.

 

Tumors arise from mutational changes to healthy cells, and are frequently deficient in one or more DNA repair pathways. The accumulation of mutations in tumor can be described by the “mutational signature”, a pattern of genetic mutations found in tumor DNA, which reflect different mutation events. Mutational signatures can be specific to certain tissues or cancer types. Many of these mutational signatures are associated with DNA repair pathways.

An in-depth study of urothelial carcinoma, which causes ~150,000 deaths annually, by Kim et al. (Nature Genetics, 2016) has identified a mutational signature in bladder cancer involving the nucleotide-excision repair (NER) pathway. Kim et al. identified a mutational signature involving ERCC2, a gene encoding a DNA helicase which plays a critical role in the NER pathway. Somatic mutations in this gene may prevent proper functioning of the NER pathway, allowing mutations to accumulate. Uniquely, urothelial cancer is the only known tumor type to date in which ERCC2 is significantly mutated.

Kim et al. used the collection of bladder carcinoma (BLCA) samples in The Cancer Genome Atlas (TCGA) to complete their analysis. Data were downloaded from the Broad Institute TCGA Genome Data Analysis Center, and samples were categorized based on mutational status. Tumors with somatic, missense mutations in ERCC2 were compared to non-mutated (wild-type) samples to identify the comprehensive mutational landscape of bladder cancer (also described in this TCGA paper).

This recipe provides a method for processing data from The Cancer Genome Atlas (TCGA), to identify samples which have mutations in specific genes. The purpose of this recipe is to categorize data by mutational status, for further downstream analysis (e.g. comparing tumors of different mutational status, etc.). Data is collected from FireBrowse; Galaxy and GenePattern are used to categorize samples by mutational status and generate GCT and CLS files. The RNA-seq datasets are gene-level normalized RSEM expression estimates.

 

TCGA Barcodes

The Cancer Genome Atlas labels its datasets with the TCGA barcode, an identifer that describes the metadata associated with sample. You can learn more about the TCGA Barcodes on the NIH National Cancer Institute Wiki page (see also: working with TCGA data).

TCGA barcodes adhere to a certain format: TCGA-00-1111-22A-33B-4444-55. For this recipe, we are interested in the Sample type, indicated by the 22A section of the barcode. For this recipe we are interested in samples with designation 01 (solid tumor, or TP) or 11 (solid tissue normal, or NT), which are paired tumor-normal samples.

Which genes are differentially expressed between my two phenotypes, based on my RNA-seq data?

This recipe provides one method to identify and visualize gene expression in different diseases and during cell differentiation and development. In collecting ChIP-seq data, we can obtain genome-wide maps of transcription factor occupancies or histone modifications between a treatment and control. In locating these regions, we can integrate ChIP-seq and RNA-seq data to better understand how these binding events regulate associated gene expression of nearby genes. An example use case of this recipe is when Laurent et al. observed how the binding of the Prep1 transcription factor influences gene regulation in mouse embryonic stem cells. The integration of both RNA-seq and Chip-seq data allows a user to identify target genes that are directly regulated by transcription factor binding or any other epigenetic occupancy in the genome.

What is Model-based Analysis of ChiP-seq (MACS)?

Model-based Analysis of ChIP-seq (MACS) is a computational algorithm that identifies genome-wide locations of transcription/chromatin factor binding or histone modifications. It is often preferred over other peak calling algorithms due to its consistency in reporting fewer false positives and its finer spatial resolution. First, it removes redundant reads to account for possible over-amplification of ChIP-DNA, which may affect peak-calling downstream. Then it shifts read positions based on the fragment size distribution to better represent the original ChIP-DNA fragment positions. Once read positions are adjusted, peak enrichment is calculated by identifying regions that are significantly enriched relative to the genomic background. MACS empirically estimates the FDR for experiments with controls for each peak, which can be used as a cutoff to filter enriched peaks. The treatment and control samples are swapped and any enriched peaks found in the control sample are regarded as false positives.

Why differential expression analysis?

We assume that most genes are not expressed all the time, but rather are expressed in specific tissues, stages of development, or under certain conditions. Genes which are expressed in one condition, such as cancerous tissue, are said to be differentially expressed when compared to normal conditions.

Use Case: ChIP-Seq and RNA-Seq Analyses Identify Components of the Wnt and Fgf Signaling Pathways as Prep1 Target Genes in Mouse Embryonic Stem Cells (Laurent et al., PLoS ONE, 2015)

The sample datatset, Series GSE6328, used for this recipe are from NCBI's GEO. We identify the interplay between epigentics and transcriptomics mouse embryonic stems cells by observing how the binding of the transcription factor, Prep1, influences gene expression. Prep1 is predominantly known for its contribution in embryonic development. In comparing genome-wide maps of mouse embryonic cells experiencing Prep1 binding to those that do not, we can identify potential target genes that are being differentially regulated by these binding events.

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