ESAT: End Sequencing Analysis Toolkit

The ESAT toolkit is designed for expression analysis of Digital expression (DGE) libraries that target transcript "ends". ESAT takes a set of alignment files (SAM or BAM) with genome alignment coordinates, a file containing transcript coordinates (BED or text file) and outputs read counts for each transcript provided.

The toolkit is implemented in Java and requires version 1.6 or higher. ESAT can be run on a single or multiple samples. When running multiple samples, all samples are processed together and the results reported as a matrix. To handle multiple samples you will need to prepare a file describing the samples indicating the alignment file locations and the experiment condition assigned to each file. (See Usage Examples below for a description of the file format.)

To download the latest version of ESAT:

The latest version of ESAT is in src/java/umms/esatJar/esat.latest.jar in the develop branch. To download the latest version, navigate to the jar file (click on src above, then java, etc.), then click on esat.jar.latest and click Download to download the binary file.

Usage:

java -jar esat.jar -task <score3p | score5p>   -alignments <sample description file> | -in <BAM/SAM alignment file>   -annotations <transcription annotation file> | -geneMapping <gene-to-transcript map file>    -out <Output file name prefix> (Additional arguments)

Argument details are provided below.

Main arguments:

Task:

-task <score3p | score5p>
Specifies the type of analysis to perform. ESAT supports two types of libraries (3' or 5'). (Default: score3p).

Annotation file:

-annotations <BED file>
Points to a transcript annotation file in BED format. If using this option, read alignment start counts will be reported for each transcript listed in the file. So, if a gene has multiple isoforms listed in this file, counts will be reported as if each transcript corresponded to a separate gene.

    OR

-geneMapping <gene-to-transcript mapping file>
Points to a gene-to-transcript mapping file whose format is described below. If using this option, all transcripts for each gene are collapsed into a single meta-transcript covering all bases contained in any transcript for that gene. This ensures that all isoforms for each gene are represented in the output. The output file references genes by the gene symbol listed in the mapping file, rather than the transcript ID. Instructions for creating a file with the correct format are provided here.

Input alignment file(s):

-in <BAM/SAM file>
For single sample runs, specifies the path to the alignment BAM/SAM file.

    OR

-alignments <sample list file>
A sample file describing all the samples to be processed. This file should contain 2 tab-separated columns, specifying one sample file per line. Column 1: Experiment/condition label (eID). All samples with the same eID will be grouped together as if all data came from the same input file. Column 2: Sample file path (BAM or SAM format). The file name must provide the full file name path, and each file must contain a header. In addition, the segments/chromosomes listed in the header must match the ones listed in the annotation or gene-to-transcript mapping file. For single-cell input data, the eID will be used as a prefix for the cell barcode, in order to identify cell barcodes from different experiments.

Output files:

-out <filename prefix>
Specifies the prefix of the output files. Output files will be named <filename prefix>.gene.txt and <filename prefix>.window.txt, containing counts of the number of reads with alignments starting within each gene and window, respectively.

Additional arguments:

Scanning window parameters:

-wLen <window length>
Length of the sliding scanning window. (Default: 50)

-wOlap <window overlap>
Overlap between adjacent sliding windows. (Default: 0)

-wExt <window extension>
Number of bases past the end (for 3’ libraries) or beginning (for 5’ libraries) of the transcript to continue the sliding windows. (Note: The actual amount of extension is truncated to prevent collision with neighboring transcripts. If the extension is truncated, a warning message is issued giving both how much the extension was truncated and the gene(s) causing the truncation.) (Default: 0)

-sigTest <p-value>
Significance test threshold for each window. A SCAN statistic p-value is computed for each window, based on the number of reads starting within the full transcript and the number of reads starting within the window. If a significance p-value is provided, only windows with a p-value less than this threshold are saved. The default setting for this parameter is 1.0, so any windows with non-zero counts are considered “significant”. If multiple significant windows abut or overlap, the range from the beginning of the first to the end of the last abutting/overlapping window is scanned with a window (of the length specified by the –wLen parameter), and the most significant window is located. Only this window is reported. (Default: 1.0)

-all
As described above, if multiple abutting/overlapping windows have significant counts, only a single, most significant, window is reported. If the –all parameter is supplied, ALL significant windows will be reported. If the –sigTest parameter is not provided, this will result in reporting counts for all windows with non-zero counts. (Default: not selected)

-unstranded
Ignores the strand of the alignment and reference, and treats all reads as if they align on the + strand. (Default: not selected)

Alignment quality check:

-quality <minimum quality>
Minimum alignment quality. Reads with alignment quality less than this threshold will not be processed. (Default: 0)

Multimapped read handling:

-multimap <method>
Indicates how multimapped reads are handled. This only applies to BAM/SAM files where the multimapped flag (in the optional fields of the alignment fields section) is provided. Multimapped reads are indicated with NH:<i>, where <i> is greater than 1. Methods:

• normal: treat multimapped reads as normal reads, receiving 1 count for each start location. This results in multiple-counting ambiguous reads. (Default)

• ignore: do not count multimapped reads. This avoids double-counting ambiguous reads.

• scale: counts each multimapped read as 1/ count, where is the number of locations to which the read is mapped (provided in the NH: field.) NOTE: Using this option greatly increases the working memory required for the program, since counts must be recorded in a floating-point array (rather than the default short integer storage). This method is mainly included for completeness, and is not recommended.

• proper: if a read is mapped to multiple genomic locations, but only one of those locations falls within the boundaries of a transcript (after splicing out introns and extension), the read is assigned to that location. If the read maps to multiple valid transcript locations, or none, it is discarded.

Low-complexity filtering:

-filtAT <n>
Remove any reads with continuous stretches of As or Ts ≥ in length. (Default: disabled)

Single-cell parameters:

-scPrep
Indicates that reads are from a single-cell protocol which uses cell barcodes and unique molecular identifiers (UMIs). The cell barcode and UMI are assumed to be attached to the read name as a colon-separated pair. (i.e., ::) (Default: disabled)

-bcMin <n>
This optional parameter specifies the minimum number of UMI-filtered reads that must be associated with a barcode to have it be considered valid. If a barcode (cell) has fewer than total UMIs, data for this barcode will not be written to the output files. (Default: 1)

-umiMin <m>
This optional parameter specifies the minimum number of times a UMI must be observed with a specific transcript for a specific cell/barcode before it is counted as valid. Each combination of cell/barcode-transcript-UMI is only counted once, but this parameter requires that combination to be seen a minimum number of times before it is counted. This helps to minimize the effect of sequencing errors in the barcode or UMI resulting in spurious transcript counts when using the UMIs to remove PCR duplicates. (Default: 1)

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Annotation BED file

This is the genome annotation file specified with the -annotations parameter, containing annotations for all transcripts being evaluated. This file must be in standard BED file format (see BED format for details.) Note that the genomic coordinates specified in this file must match those used for the alignment files.

Gene-to-transcript mapping file

This is the gene-to-transcript mapping file provided with the –geneMapping parameter, containing annotations for all transcripts being evaluated, along with the gene symbol associated with each transcript. A table containing all required information can be downloaded from the UCSC Genome Browser website (UCSC Table Browser) by selecting the species and genome assembly of interest, and the following additional fields:

• group: Genes and Gene Predictions
• track: RefSeq Genes
• table: knownGene
• output format: all fields from selected table

Then, provide an output file name and select “get output”. If the table is downloaded in gzip format, it must be un-zipped to a tab-delimited text file for input to ESAT. At a minimum, the gene mapping file must have the following (labeled) columns:

• name: transcript ID
• chrom: reference sequence chromosome (or scaffold)
• txStart: transcription start position
• txEnd: transcription end position
• name2: Gene symbol
• strand: + or - strand
• exonStarts: exon start positions
• exonEnds: exon end positions

If any of these mandatory columns are missing, ESAT will terminate with an error message. Feature coordinates are assumed to be 1-based, “right open” such that exonStart coordinates point to the base position of the start of an exon and the exonEnd coordinates point to one base after the end of the exon. (Using this coordinate system, exonEnd - exonStart = exonLength.) ExonsStarts and exonEnds are given as a matching set of comma-separated lists of values indicating the start and end position of each exon relative to txStart, similar to the blockStarts feature in BED files.

When the –geneMapping parameter is selected, all exons for all transcripts assigned to a given gene symbol are collapsed down to form a composite meta-transcript whose “exons” cover all exons of all transcripts for that gene. An example is shown below:

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Output file formats:

The gene-level output file (.gene.txt) contains a tab-delimited matrix with one row per gene/transcript provided. The first three columns contain the gene symbol or transcript ID, the chromosome, and the strand. This is followed by one column for each experiment/condition indicating the number of reads aligned to locations within the exons of the gene/transcript for that experiment/condition. The column headers for the first three columns are: “Symbol”, “chr”, and “strand”. The column headers for the remaining columns are the names of the experiments (or conditions) provided in the input file (with the –alignments parameter). If a single input file is provided with the –in parameter, the data column is named “Exp1”.

The window-level output file (.window.txt) contains a matrix with one row per window. The first 5 columns contain the gene symbol (or transcript ID), chromosome, genomic start coordinate, genomic end coordinate, and strand. This is followed by one column for each experiment/condition indicating the number of reads aligned to locations within the exonic regions contained in that window for that experiment/condition.

For both files, counts include any extension past the end of the gene/transcript specified by the -wExt parameter. Partial windows (i.e., windows with length less than the value specified with the –wLen parameter) are discarded, with one exception: if the total length of the transcript, plus any extension, is less than one full window length, read start counts are reported for all available bases for that transcript.

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Usage Examples

Processing a single .bam file using a .bed file to provide genomic coordinates of regions of interest:

  java -jar esat.jar -in sample1.bam -annotations genes.bed -out test1

This will use all default parameters (wLen=50, wExt=0, wOlap=0, 3’ scoring, stranded, no quality filtering, treating multmapped reads as normal reads, and reporting only the most significant, non-zero window within each set of adjacent non-zero-count windows), and produce two output files: test1.gene.txt and test1.window.txt.

Processing a set of .bam files using a gene-to-transcript mapping file to provide genome coordinates of transcripts:

  java –Xmx12g –jar esat.jar –alignments sampleList.txt –geneMapping geneMap.txt –wLen 200 –wExt 1000 –wOlap 50 –sigTest 0.01 –multimap ignore –out test2

The –Xmx12g switch is provided to allocate 12Gb of memory for the Java virtual machine. Alignment files are listed in the sampleList.txt file (described below), and the gene/transcript coordinates are given in the geneMap.txt file. If multiple transcripts for a gene are listed in the geneMap.txt file, all transcripts are collapsed into a single composite transcript as described in the Gene-to-transcript mapping file section above. The scanning window width is set to 200 bases with an overlap of 50 bases, and scanning extends 1000 bases past the 3’ end of the transcript. A significance test is performed for each window, windows with a p-value greater than 0.01 are discarded, and only the most significant window of length 200 within a range of overlapping “significant” windows is reported. Any reads that are marked as multimapped are skipped. Two output files, test2.gene.txt and test2.window.txt, are produced. A possible (tab-delimited) sampleList.txt file is shown below. Note that lines beginning with the comment character ‘#’ are ignored.

# Experiment 1 files:
Exp1	/home/usr1/sample1a.bam
Exp1	/home/usr1/sample1b.bam
# Experiment 2 files:
Exp2	/home/usr1/sample2a.bam
Exp2	/home/usr1/sample2b.bam

Note that multiple alignment files can be included for each experimental condition. With this example file, two columns of counts would be provided in the gene-level and window-level output files, with column headers “Exp1” and “Exp2”.

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Warning/error messages:

Extension warnings:

If the –wExt parameter is set a value greater than 0, it is possible that when attempting to extend past the end of a transcript, the extension would collide with a neighboring transcript. If this occurs, the region is extended only as far as possible without overlapping the neighboring gene, and reported with a warning such as:

WARNING … Gene ENSMUSG00000077212.1 (-) extension overlaps ENSMUSG00000042354.6_5. 400-base extension shortened to 59

This indicates that, when attempting to extend the 6th exon of the transcript ENSMUSG00000042354.6 (the “_5” indicates the zero-based exon ID) by 400 bases, it collided with the transcript for ENSMUSG00000077212.1 and had to be truncated to 59 bases in order to avoid the collision.

Isoform mismatch warnings:

When a transcript-to-gene file is provided with the –geneMapping parameter, all transcripts for each gene are collapsed into a single composite transcript, as described in the Gene-to-transcript mapping file section above. The transcripts of some genes include copies of that gene on other chromosomes, rather than just additional isoforms (splice variants) of that gene. For simplicity, the first transcript listed for each gene is assumed to have the “correct” chromosome for that gene. If transcripts of that gene listed later in the file appear on a different chromosome, they are rejected with a warning message such as:

WARNING … New isoform mismatch for Gm5512 (chr10+) with NR_002891 (chr19+)

If this causes problems for your analysis, the gene-to-transcript mapping file can be edited with this in mind. For example, if there are two known copies of gene X one two different choromsomes, and both copies are of interest to your analysis, the file could be modified such that the gene symbol for all transcripts on chromosome A are X.chrA, and all transcripts on chromosome B are named X.chrB. If it should turn out that the “true” main isoform of a gene of interest is not the first transcript listed in the file, simply modify the file to put the main isoform first.

Count overflow warnings:

The counts of the number of reads starting at each genomic location are stored in unsigned short integer (16-bit) arrays in order to minimize the memory footprint of ESAT. If the number of reads with alignments starting at a particular location exceeds the maximum possible number that can be stored (65,535), it is flagged with a warning message such as:

WARNING … location 14238898 in chr7 (-) has >65535 counts

Each overflow location is only flagged once, and counts for that location will then be saved as floating-point numbers so that the correct number of counts at that location is recorded.