QCFasta

Quality control function for high-throughput sequencing data. It takes an array of FASTQ paired or single-end reads, trims them and filters the low quality sequences. Used in whole genome (WGS), including bisulfite, or targeted (exome, RNA-seq, scRNA-seq) sequencing analyses. The function carries out the following steps:

Obtains statistics on sequences with FastQC. If input fastQCfolders from FastQC component is given, this step is skipped.
Adapter removal and quality trimming with either Trimmomatic, trimGalore or FastX.
Filters out files that do not have enough remaining sequences after the trimming step.
Obtains statistics on the preprocessed sequences with FastQC and creates html report.

Three tools are available for quality trimming: TrimGalore, Trimmomatic, and FastX. All tools remove adapters, trim low quality bases at both ends, and discard too short sequences. FastX only works with single-end data; TrimGalore and Trimmomatic can be used for both single and paired end. TrimGalore has options for RRBS libraries. Trimmomatic is faster than trimGalore and has a sliding window that is useful to trim sequences that have low quality bases that are not at the extremes of the sequence. FastX seems to be better at removing known adaptors in miRNA sets.

Most parameters work with all tools in the same way, but some have slightly different behavior (ex. stringency) or are specific to one of the tools.

QCFasta may have issues creating the quality report if the keys for the samples are single numbers from 1-10 (it is recommended to use keys that are clearly a string, i.e. not just numbers). If the fastQCfolders input is used then the parameters readkey and mateKey need to be set. These parameters are a substring of the names FastQC gave to the folders containing the reads and mates statistics. Usually reads have "_1.fq" ending while mates end in "_2.fq". In that case readKey="_1" and mateKey="_2". If you used SeqQC then the word "read" and "mate" were added to the folder names and in that case you do not need to modify readKey and mateKey since "read" and "mate" are the default values for them.

Overview of tricky parameters: More accurate descriptions of the parameters can be found in the documentation of each tool or on the individual components for Trimmomatic, TrimGalore, and Fastx (SmallRNAprep).

percent: defines the percentage of reads that need to survive the trimming step to keep the file. If more than 30% of the sample has too low quality and was discarded then perhaps you don't want to keep that file for quantifying expression. You can disable this filtering step by setting the parameter to 0.
adapterSeq: use it to define directly the string of the adapter or for Trimmomatic provide the name of the illumina adapter file (these files are included with the Trimmomatic software). If you wish to provide a different adapter for Trimmomatic save it in a file and use the adapter input; Trimmomatic does not receive directly the string in this parameter.
readKey and mateKey: these are needed to distinguish inside the folders created by fastqc between the stats for the read and the mate. Usually reads and mates are distinguished by "_1" and "_2"; but if you have something different you can set it accordingly. It is necessary a common string is used in all samples to tell reads from mates apart. In case of single-end reads do not change the default value for mateKey; readKey can be empty or set to some string common to all your filenames, but it is important that makeKey does not match anything in the output folder of FastQC.

If you add more files to your input, and then you rerun QCFasta it will restart the whole thing. Changes in the input array propagate from the beginning. Maybe this can be improved later. Changing trimming parameters for example, in principle, should not trigger a rerun of the first part (the first call to FastQC) just from the trimming steps on.

Version	5.0
Bundle	sequencing
Categories	Quality control
Authors	Alejandra Cervera (alejandra.cervera@helsinki.fi), Erkka Valo (erkka.valo@helsinki.fi)
Issue tracker	View/Report issues
Source files	component.xml function.scala
Usage	Example with default values QCFasta( reads = FASTQ, //mates = FASTQ, //fastQCfolders = BinaryFolder, //adapter = FASTA, adapterSeq = "", crop = -1, extra = "", gzip = false, headcrop = 0, isSinglecell = false, keepBothReads = false, mateKey = "mate", minLength = 20, minPercent = 20, minQuality = 20, palindromeClip = 30, percent = 0.3, qual = "", readKey = "read", simpleClip = 12, slidingWindow = "null", stringency = 2, temSwitPrimer = "GGG", threads = 2, tool = "trimmomatic", trailing = 30, umiLen = 6, umiSliding = "1:17" )

Inputs

Name	Type	Mandatory	Description
reads	Array<FASTQ>	Mandatory	An array of reads.
mates	Array<FASTQ>	Optional	An array of mates for the reads if the data is paired-end. The matching of reads to mates is done by the array keys.
fastQCfolders	Array<BinaryFolder>	Optional	If FastQC output is available you can skip running it again by providing the output folders as an array.
adapter	FASTA	Optional	Adapter file in fasta format that can only be used with trimmomatic, either single-cell or bulk.

Outputs

Name	Type	Description
qcReads	Array<FASTQ>	An array of sequences that passed the quality control step
qcMates	Array<FASTQ>	An array sequences that passed the quality control step
report	HTML	Quality control report
table	CSV	Statistics of all processed samples.
qcUnpairedReads	Array<FASTQ>	An array of sequences of which only the read passed the quality control step
qcUnpairedMates	Array<FASTQ>	An array of sequences of which only the mate passed the quality control step

Parameters

Name	Type	Default	Description
adapterSeq	string	""	Adapter specified directly as a string for TrimGalore or FastX; for Trimmomatic you can either provide the fasta file as input or specify here the Illumina adapter to use: TruSeq2-SE.fa, TruSeq2-PE.fa, TruSeq3-SE.fa, or TruSeq3-PE.fa.
crop	int	-1	Trim bases at the end of the read so it maximally has the crop size, only for Trimmomatic and FastX. The default value (-1) only works for Trimmomatic, for FastX it needs to be set to the length desired (ex. 32).
extra	string	""	Extra parameters for trim Galore! or for FastX.
gzip	boolean	false	Defines if the output sequences should be gzipped or not.
headcrop	int	0	The number of bases to remove from the start of the read. No trimming is done if the value is set to 0.
isSinglecell	boolean	false	Define if the read files are from Linnarson's single cell RNA-seq protocol (STRT).
keepBothReads	boolean	false	Defines if keep the reverse reads after read-though has been detected by palindrome mode, and the adapter sequence removed, the reverse read contains the same sequence information as the forward read, albeit in reverse complement. Only for Trimmomatic.
mateKey	string	"mate"	Key to identify mates from reads in the FastQCfolders
minLength	int	20	Reads shorter than minLength will be removed. In paired-end sequencing also the corresponding mate is removed. No trimming is done if the value is set to 0.
minPercent	int	20	Minimum percentage of bases that must have at least `minQuality` for a read to be kept. Only FastX.
minQuality	int	20	Bases below this quality threshold will be trimmed from the 5' end of the sequence.
palindromeClip	int	30	Specifies how accurate the match between the two 'adapter ligated' reads must be for PE palindrome read alignment. Only Trimmomatic.
percent	float	0.3	Percentage of good quality reads needed to keep the file.
qual	string	""	Quality version used by the sequencer (phred33 or phred64). If emtpy we use FastQC to guess the encoding.
readKey	string	"read"	Key to identify reads from mates in the FastQCfolders
simpleClip	int	12	A threshold specifies how accurate the match between any adapter must be against a read. Each matching base adds just over 0.6. Only Trimmomatic.
slidingWindow	string	"null"	Sliding window trimming where the sequence is cut if the average quality of the bases within the sliding window falls below the defined threshold. A string specifies the window size and the average required quality in the sliding window. The format is windowSize:requiredQuality. For example, 4:15 (window size = 4; required quality = 15). No trimming is done if value is set to 'null'. Only Trimmomatic.
stringency	int	2	Minimum overlap of sequence with the adapter for the bases to be trimmed (TrimGalore and FastX); allowed mismatches with the adaptor (Trimmomatic).
temSwitPrimer	string	"GGG"	minimal template-switching generated Gs
threads	int	2	Number of threads to use for the multi-threading components.
tool	string	"trimmomatic"	Choose trimmomatic, trimGalore or fastx for adapter removal and quality trimming
trailing	int	30	Remove bases from the end of the read, if quality value is below the given threshold. Only Trimmomatic.
umiLen	int	6	The length of unique molecular identifiers (UMIs), only used when isSingleCell is true.
umiSliding	string	"1:17"	slidingWindow for UMIs. For example, when UMIsliding = 1:17, any UMI bases with a quality lower than 17 will be removed. Only used when isSingleCell is true.

Test cases

Test case	Parameters▼	IN reads	IN mates	IN fastQCfolders	IN adapter	OUT qcReads	OUT qcMates	OUT report	OUT table	OUT qcUnpairedReads	OUT qcUnpairedMates
case1	properties	reads	(missing)	(missing)	(missing)	(missing)	(missing)	(missing)	(missing)	(missing)	(missing)
	readKey=_1, tool=fastx, crop=20
case2	properties	reads	(missing)	(missing)	(missing)	(missing)	(missing)	(missing)	(missing)	(missing)	(missing)
	readKey=_1, mateKey= _mates, tool=trimmomatic
case3	properties	reads	(missing)	(missing)	(missing)	(missing)	(missing)	(missing)	(missing)	(missing)	(missing)
	readKey=_1, mateKey= _mates, tool=trimGalore
case4	properties	reads	mates	(missing)	(missing)	(missing)	(missing)	(missing)	(missing)	(missing)	(missing)
	readKey =_reads, mateKey = _mates, tool=trimGalore
case5	properties	reads	mates	(missing)	(missing)	(missing)	(missing)	(missing)	(missing)	(missing)	(missing)
	readKey =_reads, mateKey = _mates, tool=trimmomatic
case6	properties	reads	mates	fastQCfolders	(missing)	(missing)	(missing)	(missing)	(missing)	(missing)	(missing)
	tool=trimmomatic

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