17 others

-v Output version of the program.


# index for reference sequences

# input reads and output
readfile2 = NULL,
type = "rna",
input_format = "gzFASTQ",
output_format = "BAM",
output_file = paste(readfile1,"subread",output_format,sep="."),

# offset value added to Phred quality scores of read bases
phredOffset = 33,

# thresholds for mapping
nsubreads = 10,
TH1 = 3,
TH2 = 1,
maxMismatches = 3,

# unique mapping and multi-mapping
unique = FALSE,
nBestLocations = 1,

# indel detection
indels = 5,
complexIndels = FALSE,

# read trimming
nTrim5 = 0,
nTrim3 = 0,

# distance and orientation of paired end reads
minFragLength = 50,
maxFragLength = 600,
PE_orientation = "fr",

# number of CPU threads
nthreads = 1,

# read group
readGroupID = NULL,
readGroup = NULL,

# read order
keepReadOrder = FALSE,
sortReadsByCoordinates = FALSE,

# color space reads
color2base = FALSE,

# dynamic programming
DP_GapOpenPenalty = -1,
DP_GapExtPenalty = 0,
DP_MismatchPenalty = 0,
DP_MatchScore = 2,

# detect structural variants
detectSV = FALSE,

# gene annotation
useAnnotation = FALSE,
annot.inbuilt = "mm39",
annot.ext = NULL,
GTF.featureType = "exon",
GTF.attrType = "gene_id",
chrAliases = NULL)

Next, we want to build an index from our reference fasta file:

cd ~/Documents/RProjects/genomics/RNA-genomics/FASTQ
source $HOME/miniconda3/bin/activate
conda activate rnaseq

subread-buildindex -o r64/r64bread.index r64/Saccharomyces_cerevisiae.R64-1-1.dna.toplevel.fa.gz

Which will create a set of files with the prefix r64bread.index (R64 is the yeast sequence version) that together comprise the index.

Now we are ready to do the alignment.

If we type subread-align into the terminal we get all the options:


## ./subread-align [options] -i -r -t -o

17.1 Mandatory arguments:

-i Base name of the index.

-r Name of an input read file. If paired-end, this should be the first read file (typically containing “R1”in the file name) and the second should be provided via “-R”. Acceptable formats include gzipped FASTQ, FASTQ, gzipped FASTA and FASTA. These formats are identified automatically.

-t Type of input sequencing data. Its values include 0: RNA-seq data 1: genomic DNA-seq data.

17.2 Optional arguments:

17.3 input reads and output

-o Name of an output file. By default, the output is in BAM format. Omitting this option makes the output be written to STDOUT.

-R Name of the second read file in paired-end data (typically containing “R2” the file name).

–SAMinput Input reads are in SAM format.

–BAMinput Input reads are in BAM format.

–SAMoutput Save mapping results in SAM format.

17.4 Phred offset

-P <3:6> Offset value added to the Phred quality score of each read base. ‘3’ for phred+33 and ‘6’ for phred+64. ‘3’ by default.

17.5 thresholds for mapping

-n Number of selected subreads, 10 by default.

-m Consensus threshold for reporting a hit (minimal number of subreads that map in consensus) . If paired-end, this gives the consensus threshold for the anchor read (anchor read receives more votes than the other read in the same pair). 3 by default

-p Consensus threshold for the non- anchor read in a pair. 1 by default.

-M Maximum number of mis-matched bases allowed in each reported alignment. 3 by default. Mis-matched bases found in soft- clipped bases are not counted.

17.6 unique mapping and multi-mapping

–multiMapping Report multi-mapping reads in addition to uniquely mapped reads. Use “-B” to set the maximum number of equally-best alignments to be reported.

-B Maximum number of equally-best alignments to be reported for a multi-mapping read. Equally-best alignments have the same number of mis-matched bases. 1 by default.

17.7 indel detection

-I Maximum length (in bp) of indels that can be detected. 5 by default. Indels of up to 200bp long can be detected.

–complexIndels Detect multiple short indels that are in close proximity (they can be as close as 1bp apart from each other).

17.8 read trimming

–trim5 Trim off number of bases from 5’ end of each read. 0 by default.

–trim3 Trim off number of bases from 3’ end of each read. 0 by default.

17.9 distance and orientation of paired end reads

-d Minimum fragment/insert length, 50bp by default.

-D Maximum fragment/insert length, 600bp by default.

-S Orientation of first and second reads, ‘fr’ by default ( forward/reverse).

17.10 number of CPU threads

-T Number of CPU threads used, 1 by default.

17.11 read group

–rg-id Add read group ID to the output.

–rg Add tag:value to the read group (RG) header in the output.

17.12 read order

–keepReadOrder Keep order of reads in BAM output the same as that in the input file. Reads from the same pair are always placed next to each other no matter this option is specified or not.

–sortReadsByCoordinates Output location-sorted reads. This option is applicable for BAM output only. A BAI index file is also generated for each BAM file so the BAM files can be directly loaded into a genome browser.

17.13 color space reads

-b Convert color-space read bases to base-space read bases in the mapping output. Note that read mapping is performed at color-space.

17.14 dynamic programming

–DPGapOpen Penalty for gap opening in short indel detection. -1 by default.

–DPGapExt Penalty for gap extension in short indel detection. 0 by default.

–DPMismatch Penalty for mismatches in short indel detection. 0 by default.

–DPMatch Score for matched bases in short indel detection. 2 by default.

17.15 detect structural variants

–sv Detect structural variants (eg. long indel, inversion, duplication and translocation) and report breakpoints. Refer to Users Guide for breakpoint reporting.

17.16 gene annotation

-a Name of an annotation file (gzipped file is accepted). GTF/GFF format by default. See -F option for more format information.

-F Specify format of the provided annotation file. Acceptable formats include ‘GTF’ (or compatible GFF format) and ‘SAF’. ‘GTF’ by default. For SAF format, please refer to Users Guide.

-A Provide a chromosome name alias file to match chr names in annotation with those in the reads. This should be a two- column comma-delimited text file. Its first column should include chr names in the annotation and its second column should include chr names in the index. Chr names are case sensitive. No column header should be included in the file.

–gtfFeature Specify feature type in GTF annotation. ‘exon’ by default. Features used for read counting will be extracted from annotation using the provided value.

–gtfAttr Specify attribute type in GTF annotation. ‘gene_id’ by default. Meta-features used for read counting will be extracted from annotation using the provided value.

17.17 others

-v Output version of the program.

Refer to Users Manual for detailed description to the arguments.

The alignment command is as follows: (I had a really hard time getting this to work by typing it into the terminal with line breaks, so wait to run it in the shell script!)

cd ~/Documents/RProjects/genomics/RNA-genomics/FASTQ
source $HOME/miniconda3/bin/activate
conda activate rnaseq

subread-align --multiMapping -t0 --sortReadsByCoordinates -a\ 
-B 3 -T 16 -i r64/r64bread.index\
-r ERR458493.fastq.gz\
-o ../BAM/ERR458493.bam &> ../BAM/R493.log

Finally, to create the count matrix we use featureCounts. The relevant options for the command are: