pipe.QuickQC: Quick Quality Control 'A to Z' Alignment & Transcriptome.

In robertdouglasmorrison/DuffyNGS: Duffy Lab NGS Analysis Pipeline for RNA-seq, DNA-seq, ChIP-seq, and RIP-seq

Quick Quality Control 'A to Z' Alignment & Transcriptome.

Description

Complete alignment pipeline for a quick QC look at a sample. Runs every pipeline step on a subset of the raw data, to generate initial assessment metrics about every stage of the alignment and transcriptome process. Useful for verifying all runtime options, sample annotation settings, needed environment variables, etc.

Usage

pipe.QuickQC(sampleID, annotationFile = "Annotation.txt", optionsFile = "Options.txt", 
		results.path = "QuickQC", banner = "QuickQC", chunkSize = 1e+06, 
		maxReads = 5e+06, pause = 0, verbose = T, nUSRkeep = 1e+06, 
		mode = c("all", "alignOnly", "pieOnly"))

Arguments

sampleID	The SampleID for this sample. This SampleID keys for one row of annotation details in the annotation file, for getting sample-specific details. The SampleID is also used as a sample-specific prefix for all files created during the processing of this sample.
annotationFile	File of sample annotation details, which specifies all needed sample-specific information about the samples under study. See DuffyNGS_Annotation.
optionsFile	File of processing options, which specifies all processing parameters that are not sample specific. See DuffyNGS_Options.
results.path	The top level folder path for writing result files to. By default, creates a new folder called 'QuickQC'.
mode	Controls the behavior of which parts of the QC pipeline get run.
banner	Character string that gets appended to the headers of all created plot images.
chunkSize	Numeric size of the buffer used for processing aligned reads. Smaller values give faster more frequent updates and QC images, but slow down overall job runtime.
maxReads	Numeric, maximum number of FASTQ reads to process. If the data is paired end, does half that many reads from each mate pair file separately.
pause	Optional delay in seconds, between plotted images, to give more time to visually inspect QC plots.
nUSRkeep	Number of Unique Short Reads (USR) to process when interrogating the 'NoHits' reads for discovery of foreign contaminants, etc.

Details

Since the full pipeline can take 10 to 36 hours to complete for one sample, that is far too long to wait to verify that all option and annotation settings are optimal. This QC pipeline is intended to give fast feedback about all aspects of the sample dataset, runtime settings, alignment success, and the user's computation environment.

Typically, with each new batch of experimental data coming off the sequencer, it is wise to run one or two samples through the QC pipe to verify that the sequencing step worked as expected. Typical things that can vary between experiments include the need for base trimming at the 5' and 3' ends, the amount of ribosomal RNA, raw read sense, etc.

Once a few QC runs have completed without error, and the transcriptomes and statistics look as expected, then all samples can confidently be submitted to the cluster as batch jobs.

Value

A large variety of subfolders and files are written to disk. Each subfolder holds a family of plot images that convey aspects of the quality of the raw FASTQ reads, the alignments, gene expression levels, and an assessment of possible foreign contaminants.

FastqReadStat	files of plot images that assess the raw base calls and quality scores in the FASTQ files. Metrics about the A/C/G/T/N distribution and variance, as well as metrics about the tile to tile variance on the slide are reported as PNG files.
AlignStats	files of plot images that assess many aspects of read alignment details from the BAM files. Metrics about: base mismatches, including location within the read and A/C/G/T/N identities; gene and chromosome coverage and distribution; insertions and deletions within the reads; Mulitply Aligned Reads (MARs); species proportions if mixed organism target; and the distribution of alignment quality scores.
transcript	transcriptome files of gene expression.
html	HTML files of the top expressing genes in each species, with hyperlinks to read pileup images that display read alignment strand calls and chromosomal coverage.
CR	'Consensus Reads' de novo assembly of No Hit reads. Raw FASTQ reads that failed to align get analyzed in a two step process: 1. grow the reads into larger 'consensus read' contigs that share overlapping DNA sequences, to create larger pieces of DNA that represent unknown material that was clearly present in the FASTQ file yet failed to align to the target genome(s); 2. Blast these DNA contigs against the giant NCBI 'NT' database, to get an unbiased call for what those DNA contigs most likely truly are. The result is text and HTML files with hyperlinks to CR pileup images, and a PNG image of the overall proportions of foreign material identified in the sample.

Note

The 'CR' step assumes there is a locally installed version of BLAST and an NT Blast database, as configured in the Options file and environment variables.

Author(s)

Bob Morrison

robertdouglasmorrison/DuffyNGS documentation built on March 24, 2024, 4:16 p.m.