De novo transcriptome reconstruction with RNA-Seq

Overview

question Questions
  • What genes are differentially expressed between G1E cells and megakaryocytes?
  • How can we generate a transcriptome de novo from RNA sequencing data?
objectives Objectives
  • Analysis of RNA sequencing data using a reference genome
  • Reconstruction of transcripts without reference transcriptome (de novo)
  • Analysis of differentially expressed genes
requirements Requirements

time Time estimation: 6 hours

Introduction

The data provided here are part of a Galaxy tutorial that analyzes RNA-seq data from a study published by Wu et al. in 2014 DOI:10.1101/gr.164830.113. The goal of this study was to investigate “the dynamics of occupancy and the role in gene regulation of the transcription factor Tal1, a critical regulator of hematopoiesis, at multiple stages of hematopoietic differentiation.” To this end, RNA-seq libraries were constructed from multiple mouse cell types including G1E - a GATA-null immortalized cell line derived from targeted disruption of GATA-1 in mouse embryonic stem cells - and megakaryocytes. This RNA-seq data was used to determine differential gene expression between G1E and megakaryocytes and later correlated with Tal1 occupancy. This dataset (GEO Accession: GSE51338) consists of biological replicate, paired-end, poly(A) selected RNA-seq libraries. Because of the long processing time for the large original files, we have downsampled the original raw data files to include only reads that align to chromosome 19 and a subset of interesting genomic loci identified by Wu et al.

Analysis strategy

The goal of this exercise is to identify what transcripts are present in the G1E and megakaryocyte cellular states and which transcripts are differentially expressed between the two states. We will use a de novo transcript reconstruction strategy to infer transcript structures from the mapped reads in the absence of the actual annotated transcript structures. This will allow us to identify novel transcripts and novel isoforms of known transcripts, as well as identify differentially expressed transcripts.

Agenda

In this tutorial, we will deal with:

  1. Analysis strategy
    1. Data upload
    2. Quality control
  2. Mapping
  3. De novo transcript reconstruction
  4. Transcriptome assembly
  5. Analysis of the differential gene expression
    1. Count the number of reads per transcript
    2. Perform differential gene expression testing
  6. Visualization

Data upload

Due to the large size of this dataset, we have downsampled it to only include reads mapping to chromosome 19 and certain loci with relevance to hematopoeisis. This data is available at Zenodo, where you can find the forward and reverse reads corresponding to replicate RNA-seq libraries from G1E and megakaryocyte cells and an annotation file of RefSeq transcripts we will use to generate our transcriptome database.

hands_on Hands-on: Data upload

  1. Create a new history for this RNA-seq exercise
  2. Open the data upload manager (Get Data -> Upload file)
  3. Copy and paste the links for the reads and annotation file
  4. Select Paste/Fetch Data
  5. Paste the link(s) into the text field
  6. Change the datatype of the read files to fastqsanger
  7. Change the datatype of the annotation file to gtf and assign the Genome as mm10
  8. Press Start
  9. Rename the files in your history to retain just the necessary information (e.g. “G1E R1 forward reads”)

    Data available from zenodo: DOI

    Below are the links to the read files that can be copied and pasted in the upload manager.

    https://zenodo.org/record/583140/files/G1E_rep1_forward_read_%28SRR549355_1%29
    https://zenodo.org/record/583140/files/G1E_rep1_reverse_read_%28SRR549355_2%29
    https://zenodo.org/record/583140/files/G1E_rep2_forward_read_%28SRR549356_1%29
    https://zenodo.org/record/583140/files/G1E_rep2_reverse_read_%28SRR549356_2%29
    https://zenodo.org/record/583140/files/Megakaryocyte_rep1_forward_read_%28SRR549357_1%29
    https://zenodo.org/record/583140/files/Megakaryocyte_rep1_reverse_read_%28SRR549357_2%29
    https://zenodo.org/record/583140/files/Megakaryocyte_rep2_forward_read_%28SRR549358_1%29
    https://zenodo.org/record/583140/files/Megakaryocyte_rep2_reverse_read_%28SRR549358_2%29
    https://zenodo.org/record/583140/files/RefSeq_reference_GTF_%28DSv2%29
    

    You will need to fetch the link to the annotation file yourself ;)

Quality control

For quality control, we use similar tools as described in NGS-QC tutorial: FastQC and Trimmomatic.

hands_on Hands-on: Quality control

  1. FastQC tool: Run FastQC on the forward and reverse read files to assess the quality of the reads.

    question Questions

    1. What is the read length?
    2. Is there anything interesting about the quality of the base calls based on the position in the reads?

    solution Solution

    1. The read length is 99 bp
    2. The quality of base calls declines throughout a sequencing run.
  2. Trimmomatic tool: Trim off the low quality bases from the ends of the reads to increase mapping efficiency. Run Trimmomatic on each pair of forward and reverse reads with the following settings:

    • “Single-end or paired-end reads?”: Paired-end (two separate input files)
      • param-file “Input FASTQ file (R1/first of pair)”: G1E forward read (R1)
      • param-file “Input FASTQ file (R2/second of pair)”: G1E reverse read (R1)
    • “Perform initial ILLUMINACLIP step?”: No
  3. FastQC tool: Re-run FastQC on trimmed reads and inspect the differences.

    question Questions

    1. What is the read length?
    2. Is there anything interesting about the quality of the base calls based on the position in the reads?

    solution Solution

    1. The read lengths range from 1 to 99 bp after trimming
    2. The average quality of base calls does not drop off as sharply at the 3’ ends of reads.

    Before and after trimming comparison

Now that we have trimmed our reads and are fortunate that there is a reference genome assembly for mouse, we will align our trimmed reads to the genome.

comment Comment

Instead of running a single tool multiple times on all your data, would you rather run a single tool on multiple datasets at once? Check out the dataset collections feature of Galaxy!

Mapping

To make sense of the reads, their positions within mouse genome must be determined. This process is known as aligning or ‘mapping’ the reads to the reference genome.

comment Comment

Do you want to learn more about the principles behind mapping? Follow our training

In the case of a eukaryotic transcriptome, most reads originate from processed mRNAs lacking introns. Therefore, they cannot be simply mapped back to the genome as we normally do for reads derived from DNA sequences. Instead, the reads must be separated into two categories:

Spliced mappers have been developed to efficiently map transcript-derived reads against genomes. HISAT is an accurate and fast tool for mapping spliced reads to a genome. Another popular spliced aligner is TopHat, but we will be using HISAT in this tutorial.

comment Comment

As it is sometimes quite difficult to determine which settings correspond to those of other programs, the following table might be helpful to identify the library type:

Library type Infer Experiment TopHat HISAT htseq-count featureCounts
PE 1++,1–,2+-,2-+ FR Second Strand FR yes 1
PE 1+-,1-+,2++,2– FR First Strand RF reverse 2
SE ++,– FR Second Strand F yes 1
SE +-,-+ FR First Strand R reverse 2
SE,PE undecided FR Unstranded default no 0

hands_on Hands-on: Spliced mapping

  1. HISAT2 tool: Run HISAT2 on one forward/reverse read pair and modify the following settings:
    • “Source for the reference genome”: Use a built-in genome
      • param-file “Select a reference genome”: Mouse (Mus Musculus): mm10
    • “Single-end or paired-end reads?”: Paired-end
      • param-file “FASTA/Q file #1”: trimmed G1E forward read (R1)
      • param-file “FASTA/Q file #2”: trimmed G1E reverse read (R1)
      • “Specify strand information”: Forward(FR)
    • “Advanced options”
      • “Spliced alignment options”
        • “Penalty for non-canonical splice sites”: ‘3’
        • “Penalty function for long introns with canonical splice sites”: ‘Constant [f(x) = B]’’
        • “Constant term (B)”: ‘0.0’
        • “Penalty function for long introns with non-canonical splice sites”: ‘Constant [f(x) = B]’’
        • “Transcriptome assembly reporting”: ‘Report alignments tailored for transcript assemblers including StringTie’
  2. HISAT2 tool: Run HISAT2 on the remaining forward/reverse read pairs with the same parameters.

De novo transcript reconstruction

Now that we have mapped our reads to the mouse genome with HISAT, we want to determine transcript structures that are represented by the aligned reads. This is called de novo transcriptome reconstruction. This unbiased approach permits the comprehensive identification of all transcripts present in a sample, including annotated genes, novel isoforms of annotated genes, and novel genes. While common gene/transcript databases are quite large, they are not comprehensive, and the de novo transcriptome reconstruction approach ensures complete transcriptome(s) identification from the experimental samples. The leading tool for transcript reconstruction is Stringtie. Here, we will use Stringtie to predict transcript structures based on the reads aligned by HISAT.

hands_on Hands-on: Transcriptome reconstruction

  1. Stringtie tool: Run Stringtie on the HISAT2 alignments using the default parameters.
    • Use batch mode to run all four samples from one tool form.
    • param-file “Specify strand information”: Forward (FR)

Transcriptome assembly

We just generated four transcriptomes with Stringtie representing each of the four RNA-seq libraries we are analyzing. Since these were generated in the absence of a reference transcriptome, and we ultimately would like to know what transcript structure corresponds to which annotated transcript (if any), we have to make a transcriptome database. We will use the tool Stringtie - Merge to combine redundant transcript structures across the four samples and the RefSeq reference. Once we have merged our transcript structures, we will use GFFcompare to annotate the transcripts of our newly created transcriptome so we know the relationship of each transcript to the RefSeq reference.

hands_on Hands-on: Transcriptome assembly

  1. Stringtie-merge tool: Run Stringtie-merge on the Stringtie assembled transcripts along with the RefSeq annotation file we imported earlier.
    • param-file “input_gtf”: all four Stringtie assemblies
    • param-file “guide_gff”: RefSeq GTF mm10
  2. GFFCompare tool: Run GFFCompare on the Stringtie-merge generated transcriptome along with the RefSeq annotation file.
    • param-file “GTF inputs for comparison”: output of Stringtie-merge
    • “Use Reference Annotation”: Yes
      • param-file “Reference Annotation”: RefSeq GTF mm10
    • “Use Sequence Data”: Yes
      • “Choose the source for the reference list”: Locally cached
        • “Using reference genome”: ‘Mouse (Mus Musculus): mm10’

comment Note: Transcript categorization used by GFFcompare

Class code Transcript category
= Annotated in reference
j Novel isoform of reference
u Intergenic
x Anti-sense
r Repetitive
c Contained in exon of reference
s Anti-sense spliced intronic
e Single exon transfrag overlapping a reference exon and at least 10 bp of a reference intron, indicating a possible pre-mRNA fragment.
i A transfrag falling entirely within a reference intron
o Generic exonic overlap with a reference transcript
p Possible polymerase run-on fragment (within 2Kbases of a reference transcript)

Analysis of the differential gene expression

We just generated a transriptome database that represents the transcripts present in the G1E and megakaryocytes samples. This database provides the location of our transcripts with non-redundant identifiers, as well as information regarding the origin of the transcript.

We now want to identify which transcripts are differentially expressed between the G1E and megakaryocyte cellular states. To do this we will implement a counting approach using FeatureCounts to count reads per transcript. Then we will provide this information to DESeq2 to generate normalized transcript counts (abundance estimates) and significance testing for differential expression.

Count the number of reads per transcript

To compare the abundance of transcripts between different cellular states, the first essential step is to quantify the number of reads per transcript. FeatureCounts is one of the most popular tools for counting reads in genomic features. In our case, we’ll be using FeatureCounts to count reads aligning in exons of our GFFCompare generated transcriptome database.

The recommended mode is “union”, which counts overlaps even if a read only shares parts of its sequence with a genomic feature and disregards reads that overlap more than one feature.

hands_on Hands-on: Counting the number of reads per transcript

  1. FeatureCounts tool: Run FeatureCounts on the aligned reads (HISAT2 output) using the GFFCompare transcriptome database as the annotation file.

    • param-file “Alignment file”: four HISAT2 aligned read files
    • “Specify strand information”: Stranded (Forward)
    • param-file “Gene annotation file”: in your history
      • “Gene annotation file”: the annotated transcripts GTF file output by GFFCompare
    • “Advanced options”
      • “GFF gene identifier”: transcript_id

Perform differential gene expression testing

Transcript expression is estimated from read counts, and attempts are made to correct for variability in measurements using replicates. This is absolutely essential to obtaining accurate results. We recommend having at least two biological replicates.

DESeq2 is a great tool for differential gene expression analysis. It accepts read counts produced by FeatureCounts and applies size factor normalization:

hands_on Hands-on:

  1. DESeq2 tool: Run DESeq2 with the following parameters:
    • “1: Factor”
      • “1: Factor level”: G1E
        • param-file “Counts file(s)”: featureCount files corresponding to the two G1E replicates
      • “2: Factor level”: Mega
        • param-file “Counts file(s)”: featureCount files corresponding to the two Mega replicates

      comment Comment

      You can select several files by holding down the CTRL (or COMMAND) key and clicking on the desired files

    • “Visualising the analysis results”: Yes
    • “Output normalized counts table”: Yes

The first output of DESeq2 is a tabular file. The columns are:

  1. Gene identifiers
  2. Mean normalized counts, averaged over all samples from both conditions
  3. Logarithm (base 2) of the fold change (the values correspond to up- or downregulation relative to the condition listed as Factor level 1)
  4. Standard error estimate for the log2 fold change estimate
  5. Wald statistic
  6. p-value for the statistical significance of this change
  7. p-value adjusted for multiple testing with the Benjamini-Hochberg procedure which controls false discovery rate (FDR)

hands_on Hands-on:

  1. Filter tool: Run Filter to extract genes with a significant change in gene expression (adjusted p-value less than 0.05) between treated and untreated samples

    question Question

    How many transcripts have a significant change in expression between these conditions?

    solution Solution

    To filter, use “c7<0.05”. And we get 249 transcripts with a significant change in gene expression between the G1E and megakaryocyte cellular states.

  2. Filter tool: Determine how many transcripts are up or down regulated in the G1E state.

    comment Comments

    Rename your datasets for the downstream analyses

    question Question

    Are there more upregulated or downregulated genes in the treated samples?

    solution Solution

    To obtain the up-regulated genes in the G1E state, we filter the previously generated file (with the significant change in transcript expression) with the expression “c3>0” (the log2 fold changes must be greater than 0). We obtain 102 genes (40.9% of the genes with a significant change in gene expression). For the down-regulated genes in the G1E state, we did the inverse and we find 149 transcripts (59% of the genes with a significant change in transcript expression).

In addition to the list of genes, DESeq2 outputs a graphical summary of the results, useful to evaluate the quality of the experiment:

  1. Histogram of p-values for all tests

    DESeq2 histogram

  2. MA plot: global view of the relationship between the expression change of conditions (log ratios, M), the average expression strength of the genes (average mean, A), and the ability of the algorithm to detect differential gene expression. The genes that passed the significance threshold (adjusted p-value < 0.1) are colored in red.

    MA plot

  3. Principal Component Analysis (PCA) and the first two axes

    Principal component analysis output

    Each replicate is plotted as an individual data point. This type of plot is useful for visualizing the overall effect of experimental covariates and batch effects.

  4. Heatmap of sample-to-sample distance matrix: overview over similarities and dissimilarities between samples

    Heatmap of sample-to-sample distance matrix

  5. Dispersion estimates: gene-wise estimates (black), the fitted values (red), and the final maximum a posteriori estimates used in testing (blue)

    Dispersion estimates

    This dispersion plot is typical, with the final estimates shrunk from the gene-wise estimates towards the fitted estimates. Some gene-wise estimates are flagged as outliers and not shrunk towards the fitted value. The amount of shrinkage can be more or less than seen here, depending on the sample size, the number of coefficients, the row mean and the variability of the gene-wise estimates.

For more information about DESeq2 and its outputs, you can have a look at DESeq2 documentation.

Visualization

Now that we have a list of transcript expression levels and their differential expression levels, it is time to visually inspect our transcript structures and the reads they were predicted from. It is a good practice to visually inspect (and present) loci with transcripts of interest. Fortunately, there is a built-in genome browser in Galaxy, Trackster, that make this task simple (and even fun!).

In this last section, we will convert our aligned read data from BAM format to bigWig format to simplify observing where our stranded RNA-seq data aligned to. We’ll then initiate a session on Trackster, load it with our data, and visually inspect our interesting loci.

hands_on Hands-on: Converting aligned read files to bigWig format

  1. bamCoverage tool: Run bamCoverage on all four aligned read files (HISAT2 output) with the following parameters:
    • “Bin size in bases”: ‘1’
    • “Effective genome size”: ‘mm9 (2150570000)’’
    • “Advanced options”
      • “Only include reads originating from fragments from the forward or reverse strand”: ‘forward’
  2. Rename tool: Rename the outputs to reflect the origin of the reads and that they represent the reads mapping to the PLUS strand.

  3. bamCoverage tool: Repeat Step 1 except changing the following parameter:
    • “Only include reads originating from fragments from the forward or reverse strand”: ‘reverse’
  4. Rename tool: Rename the outputs to reflect the origin of the reads and that they represent the reads mapping to the MINUS strand.

hands_on Hands-on: Trackster based visualization

  1. Viz tool: On the center console at the top of the Galaxy interface, choose “ Visualization” -> “New track browser”
    • Name your visualization someting descriptive under “Browser name:”
    • Choose “Mouse Dec. 2011 (GRCm38/mm10) (mm10)” as the “Reference genome build (dbkey)
    • Click “Create” to initiate your Trackster session Trackster opening window
  2. Viz tool: Click “Add datasets to visualization”
    • Select the “RefSeq GTF mm10” file
    • Select the output files from Stringtie
    • Select the output file from GFFCompare
    • Select the output files from bamCoverage
  3. tool: Using the grey labels on the left side of each track, drag and arrange the track order to your preference.

  4. tool: Hover over the grey label on the left side of the “RefSeq GTF mm10” track and click the “Edit settings” icon.
    • Adjust the block color to blue (#0000ff) and antisense strand color to red (#ff0000)
  5. tool: Repeat the previous step on the output files from StringTie and GFFCompare.

  6. tool: Hover over the grey label on the left side of the “G1E R1 plus” track and click the “Edit settings” icon.
    • Adjust the color to blue (#0000ff)
  7. tool: Repeat the previous step on the other three bigWig files representing the plus strand.

  8. tool: Hover over the grey label on the left side of the “G1E R1 minus” track and click the “Edit settings” icon.
    • Adjust the color to red (#ff0000)
  9. tool: Repeat the previous step on the other three bigWig files representing the minus strand.

  10. tool: Adjust the track height of the bigWig files to be consistent for each set of plus strand and minus strand tracks. Adjusting the track height
  11. tool: Direct Trackster to the coordinates: chr11:96191452-96206029, what do you see?

question Question

what do you see?

solution Solution

  1. There are two clusters of transcripts that are exclusively expressed in the G1E background
  2. The left-most transcript is the Hoxb13 transcript
  3. The center cluster of transcripts are not present in the RefSeq annotation and are determined by GFFCompare to be “u” and “x”

Conclusion

In this tutorial, we have analyzed RNA sequencing data to extract useful information, such as which genes are expressed in the G1E and megakaryocyte cellular states and which of these genes are differentially expressed between the two cellular states. In addition, we identified unannotated genes that are expressed in a cell-state dependent manner and at a locus with relevance to differentiation and development. To identify these transcripts, we analyzed RNA sequence datasets using a de novo transcriptome reconstruction RNA-seq data analysis approach. This approach can be summed up with the following scheme:

RNAseq de novo tutorial workflow

keypoints Key points

  • De novo transcriptome reconstruction is the ideal approach for identifying differentially expressed known and novel transcripts.
  • Differential gene expression testing is improved with the use of replicate experiments and deep sequence coverage.
  • Visualizing data on a genome browser is a great way to display interesting patterns of differential expression.

Useful literature

Further information, including links to documentation and original publications, regarding the tools, analysis techniques and the interpretation of results described in this tutorial can be found here.

congratulations Congratulations on successfully completing this tutorial!