Introduction
Modern sequencing techniques enable researchers to sequence the genome at greater depth than ever before. NGS stands fro Next Generation Sequencing and allows for extremely high throughput, scalability, and speed. Using this technology, it is possible to determine nucleotide sequences within entire genomes or specific regions of DNA or RNA.
All high-throughput sequencing analyses require quality checks and processing of reads. Before we can quantify the genomic signal of interest and apply statistical and/or machine learning methods, we must process, quality check, and align the millions of reads generated by these sequencing experiments.
In this tutorial, we will assess the read quality and processing which are fundamental steps in all high-throughput sequencing analyses.
Methods
1. Set working directory
setwd("C:/Users/manso/OneDrive - University of West London/MSc Bioinformatics - UWL/6.BGA - Bioinformatics and Genome Analysis/week 2 - NGS data QC and alignment in R/practical")
2. Install necessary packages and load libraries
if (!requireNamespace("BiocManager", quietly = TRUE))
install.packages("BiocManager")
BiocManager::install("Rsubread")
BiocManager::install("ShortRead")
BiocManager::install("fastqcr")
BiocManager::install("Rqc")
BiocManager::install("QuasR")
BiocManager::install("ngsReports")
3. Download Fasq data
We will use system.file() to upload data into a folder we will call ‘mydata’ in.
mydata = system.file(package="ShortRead", "extdata/E-MTAB-1147")
Now feed fastq.qz files in “mydata” to the quality check function rqc() to create the object qcRes:
qcRes=rqc(path = mydata, pattern = ".fastq.gz", openBrowser=FALSE)
Report is in C:/Users/manso/AppData/Local/Temp/RtmpMbKuwN/rqc_report.html Pasting the file path in browser window will show the html report.
4. Plot quality metrics
Plot a sequence quality per base/cycle metrics for our fastq files in qcRes object use
rqcCycleQualityBoxPlot(qcRes)
What does this graph depicts?
The graph above shows the sequence quality per base/cycle metrics, we can observe that the quality of the reads degrades at the end which is expected for long sequences. Good samples will have median quality scores per base above 28. If scores are below 20 towards the ends, you can think about trimming the reads.
Why is the x-axis labelled as ‘cycle’?
Cycles correspond to bases/nucleotides along the read, and the number of cycles is equivalent to the read length.
5. Find Sequence content per base/cycle
rqcCycleBaseCallsLinePlot(qcRes)
What does the per base sequence content show?
Per-base sequence content shows nucleotide proportions for each position.
This plot shows sequence bias – what is the probable cause of the bias?
Some types of sequencing libraries can produce a biased sequence composition. For example, in RNA-Seq, it is common to have bias at the beginning of the reads. This happens because of random primers annealing to the start of reads during RNA-Seq library preparation. These primers are not truly random, which leads to a variation at the beginning of the reads. Although RNA-seq experiments will usually have these biases, this will not affect the ability of measuring gene expression.
6. Filtering and trimming read
It might be necessary to trim or filter the reads based on the quality check results. It may be that adapters are present on either side of the read, or that technical errors are causing the base quality to diminish toward the ends of the read. This portion of the read should be trimmed, in both cases, to allow alignment or better alignment of the genome.
We can use QuasR or ShortRead packages to trim and filter reads the reads. However, QuasR::preprocessReads() function provides a single interface to multiple pre-processing.
6.1. First, Obtain a list of fastq file paths:
fastqFiles <- system.file(package="ShortRead",
"extdata/E-MTAB-1147",
c("ERR127302_1_subset.fastq.gz", "ERR127302_2_subset.fastq.gz"))
For this tutorial we are using built in training data from the package - and we access this data using ‘system.file’ command. When using our own data we must create the correct filepath for our data.
6.2. Next, Define processed fastq file names:
6.3. Then we Process fastq files with the following parameters as an example:
- Remove reads that have more than 1 N, (nBases)
- trim 3 bases from the end of the reads (truncateEndBases)
- remove ACCCGGGA patern if it occurs at the start (Lpattern)
- remove reads shorter than 40 base-pairs (minLength)
preprocessReads(fastqFiles, outfiles, nBases=1,
truncateEndBases=3,Lpattern="ACCCGGGA",
minLength=40)
ERR127302_1_subset.fastq.gz
totalSequences 20000
matchTo5pAdapter 3804
matchTo3pAdapter 0
tooShort 355
tooManyN 4
lowComplexity 0
totalPassed 19641
ERR127302_2_subset.fastq.gz
totalSequences 20000
matchTo5pAdapter 3398
matchTo3pAdapter 0
tooShort 426
tooManyN 1
lowComplexity 0
totalPassed 195736.4. Filter out reads with quality score below 20:
- Obtain a list of fastq file paths:
fastqFile <- system.file(package="ShortRead",
"extdata/E-MTAB-1147",
"ERR127302_1_subset.fastq.gz")
- Read fastq file:
fq = readFastq(fastqFile)
- Get quality scores per base as a matrix:
qPerBase = as(quality(fq), "matrix")
- Get the number of bases per read that have quality score below 20:
qcount = rowSums( qPerBase <= 20)
- Number of reads where all Phred scores >= 20
fq[qcount == 0]
class: ShortReadQ
length: 10699 reads; width: 72 cycles- Write out fastq file with only reads where all quality scores per base are above 20:
writeFastq(fq[qcount == 0], paste(fastqFile, "Qfiltered", sep="_"))
ShortRead::FastqStreamer() - streams the fastq file in blocks, each with a pre-defined number of reads. Which can be accessed using the yield() function. As we call the yield() function after opening a fastq file with FastqStreamer(), a new section of the file is read into memory.
- Set up a streaming with block size 1000. Every time you call the yield() function 1000 read portion of the file will be read successively.
f <- FastqStreamer(fastqFile, readerBlockSize=1000)
- Set up a while loop to call yield() function to go through the file:
This loop will remove reads where all quality scores are < 20, get quality scores per base as a matrix; get number of bases per read that have Q score < 20, write fastq file with mode=“a”, to every new block, and written out to the same file.
7. Map or align the reads to the genome (library(QuasR) is needed)
7.1. Copy example data to current working directory:
file.copy(system.file(package="QuasR", "extdata"), ".", recursive=TRUE)
[1] TRUE7.2. Get genome file in fasta format:
genomeFile <- "extdata/hg19sub.fa"
7.3. Get text file containing sample names and fastq file paths:
sampleFile <- "extdata/samples_chip_single.txt"
7.4. Create alignments:
proj <- qAlign(sampleFile, genomeFile)
With qAlign(), alignment parameters are automatically selected based on different types of alignment problems
8. Map or align the reads to the genome with Rsubread
Data files needed:
Mouse chromosome
1 Rsubread index files (~400MB);
Targets2.txt
The 12 fastq.gz files for the mouse dataset: 12 fastq files with 1000 reads each
4 index files for chr 1 for mm10, targets files with sample information.
- Put all the sequencing read data (.fastq.gz files) in your data directory.
- Find the files and command Rsubread aligner which files to look at.
8.1. Search for all .fastq.gz files in the directory using the list.files command.
fastq.files <- list.files(path = "C:/Users/manso/OneDrive - University of West London/MSc Bioinformatics - UWL/6.BGA - Bioinformatics and Genome Analysis/week 2 - NGS data QC and alignment in R/practical/MouseMamRNA-Seq", pattern = ".fastq.gz$", full.names = TRUE)
fastq.files
[1] "C:/Users/manso/OneDrive - University of West London/MSc Bioinformatics - UWL/6.BGA - Bioinformatics and Genome Analysis/week 2 - NGS data QC and alignment in R/practical/MouseMamRNA-Seq/SRR1552444.fastq.gz"
[2] "C:/Users/manso/OneDrive - University of West London/MSc Bioinformatics - UWL/6.BGA - Bioinformatics and Genome Analysis/week 2 - NGS data QC and alignment in R/practical/MouseMamRNA-Seq/SRR1552445.fastq.gz"
[3] "C:/Users/manso/OneDrive - University of West London/MSc Bioinformatics - UWL/6.BGA - Bioinformatics and Genome Analysis/week 2 - NGS data QC and alignment in R/practical/MouseMamRNA-Seq/SRR1552446.fastq.gz"
[4] "C:/Users/manso/OneDrive - University of West London/MSc Bioinformatics - UWL/6.BGA - Bioinformatics and Genome Analysis/week 2 - NGS data QC and alignment in R/practical/MouseMamRNA-Seq/SRR1552447.fastq.gz"
[5] "C:/Users/manso/OneDrive - University of West London/MSc Bioinformatics - UWL/6.BGA - Bioinformatics and Genome Analysis/week 2 - NGS data QC and alignment in R/practical/MouseMamRNA-Seq/SRR1552448.fastq.gz"
[6] "C:/Users/manso/OneDrive - University of West London/MSc Bioinformatics - UWL/6.BGA - Bioinformatics and Genome Analysis/week 2 - NGS data QC and alignment in R/practical/MouseMamRNA-Seq/SRR1552449.fastq.gz"
[7] "C:/Users/manso/OneDrive - University of West London/MSc Bioinformatics - UWL/6.BGA - Bioinformatics and Genome Analysis/week 2 - NGS data QC and alignment in R/practical/MouseMamRNA-Seq/SRR1552450.fastq.gz"
[8] "C:/Users/manso/OneDrive - University of West London/MSc Bioinformatics - UWL/6.BGA - Bioinformatics and Genome Analysis/week 2 - NGS data QC and alignment in R/practical/MouseMamRNA-Seq/SRR1552451.fastq.gz"
[9] "C:/Users/manso/OneDrive - University of West London/MSc Bioinformatics - UWL/6.BGA - Bioinformatics and Genome Analysis/week 2 - NGS data QC and alignment in R/practical/MouseMamRNA-Seq/SRR1552452.fastq.gz"
[10] "C:/Users/manso/OneDrive - University of West London/MSc Bioinformatics - UWL/6.BGA - Bioinformatics and Genome Analysis/week 2 - NGS data QC and alignment in R/practical/MouseMamRNA-Seq/SRR1552453.fastq.gz"
[11] "C:/Users/manso/OneDrive - University of West London/MSc Bioinformatics - UWL/6.BGA - Bioinformatics and Genome Analysis/week 2 - NGS data QC and alignment in R/practical/MouseMamRNA-Seq/SRR1552454.fastq.gz"
[12] "C:/Users/manso/OneDrive - University of West London/MSc Bioinformatics - UWL/6.BGA - Bioinformatics and Genome Analysis/week 2 - NGS data QC and alignment in R/practical/MouseMamRNA-Seq/SRR1552455.fastq.gz"The pattern argument takes a regular expression. In this case, we are using the “$” to mean the end of the string, so we will only get files that end in “.fastq.gz”
8.2. Build the Index:
#buildindex(basename="./reference_index",reference=ref)
buildindex(basename="chr1_mm10",reference="chr1.fa")
========== _____ _ _ ____ _____ ______ _____
===== / ____| | | | _ \| __ \| ____| /\ | __ \
===== | (___ | | | | |_) | |__) | |__ / \ | | | |
==== \___ \| | | | _ <| _ /| __| / /\ \ | | | |
==== ____) | |__| | |_) | | \ \| |____ / ____ \| |__| |
========== |_____/ \____/|____/|_| \_\______/_/ \_\_____/
Rsubread 2.8.2
//================================= setting ==================================\\
|| ||
|| Index name : chr1_mm10 ||
|| Index space : base space ||
|| Index split : no-split ||
|| Repeat threshold : 100 repeats ||
|| Gapped index : no ||
|| ||
|| Free / total memory : 16.2GB / 31.4GB ||
|| ||
|| Input files : 1 file in total ||
|| o chr1.fa ||
|| ||
\\============================================================================//
//================================= Running ==================================\\
|| ||
|| Check the integrity of provided reference sequences ... ||
|| No format issues were found ||
|| Scan uninformative subreads in reference sequences ... ||
|| 41288 uninformative subreads were found. ||
|| These subreads were excluded from index building. ||
|| Estimate the index size... ||
|| 8%, 0 mins elapsed, rate=6107.6k bps/s ||
|| 16%, 0 mins elapsed, rate=5781.8k bps/s ||
|| 24%, 0 mins elapsed, rate=5682.1k bps/s ||
|| 33%, 0 mins elapsed, rate=5615.0k bps/s ||
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|| 83%, 0 mins elapsed, rate=5514.6k bps/s ||
|| 91%, 0 mins elapsed, rate=5513.6k bps/s ||
|| 4.0 GB of memory is needed for index building. ||
|| Build the index... ||
|| 8%, 0 mins elapsed, rate=2291.0k bps/s ||
|| 16%, 0 mins elapsed, rate=2356.7k bps/s ||
|| 24%, 0 mins elapsed, rate=2372.1k bps/s ||
|| 33%, 0 mins elapsed, rate=2375.6k bps/s ||
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|| 49%, 0 mins elapsed, rate=2383.1k bps/s ||
|| 58%, 0 mins elapsed, rate=2388.2k bps/s ||
|| 66%, 0 mins elapsed, rate=2390.2k bps/s ||
|| 74%, 1 mins elapsed, rate=2391.7k bps/s ||
|| 83%, 1 mins elapsed, rate=2391.0k bps/s ||
|| 91%, 1 mins elapsed, rate=2390.5k bps/s ||
|| Save current index block... ||
|| [ 0.0% finished ] ||
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|| ||
|| Total running time: 3.0 minutes. ||
||Index C:\Users\manso\OneDrive - University of West London\MSc Bioinfor ... ||
|| ||
\\============================================================================//With our index generated, we can align the reads with the align command.
According to align’s default settings, it only keeps reads that uniquely map to the reference genome. We need this when testing differential gene expression, because each read is unambiguously assigned to one place in the genome, making interpretation of results easier.
8.3. Align the reads using the align command:
#to view and change parameters run: args(align)
align("chr1_mm10", readfile1 = fastq.files,
useAnnotation=T, nthreads=10, annot.inbuilt="mm10",
phredOffset = 64 )
========== _____ _ _ ____ _____ ______ _____
===== / ____| | | | _ \| __ \| ____| /\ | __ \
===== | (___ | | | | |_) | |__) | |__ / \ | | | |
==== \___ \| | | | _ <| _ /| __| / /\ \ | | | |
==== ____) | |__| | |_) | | \ \| |____ / ____ \| |__| |
========== |_____/ \____/|____/|_| \_\______/_/ \_\_____/
Rsubread 2.8.2
//================================= setting ==================================\\
|| ||
|| Function : Read alignment (RNA-Seq) ||
|| Input file : SRR1552444.fastq.gz ||
|| Output file : SRR1552444.fastq.gz.subread.BAM (BAM) ||
|| Index name : chr1_mm10 ||
|| ||
|| ------------------------------------ ||
|| ||
|| Threads : 10 ||
|| Phred offset : 64 ||
|| Min votes : 3 / 10 ||
|| Max mismatches : 3 ||
|| Max indel length : 5 ||
|| Report multi-mapping reads : yes ||
|| Max alignments per multi-mapping read : 1 ||
|| Annotations : inbuilt (mm10) ||
|| ||
\\============================================================================//
//================ Running (01-May-2022 18:26:38, pid=23920) =================\\
|| ||
|| Check the input reads. ||
|| The input file contains base space reads. ||
|| Initialise the memory objects. ||
|| Estimate the mean read length. ||
|| WARNING - The specified Phred score offset (64) seems incorrect. ||
|| ASCII values of the quality scores of read bases included in ||
|| the first 999 reads were found to be within the range of ||
|| [35,74]. ||
|| ||
|| Create the output BAM file. ||
|| Check the index. ||
|| Init the voting space. ||
|| Load the annotation file. ||
|| 222996 annotation records were loaded. ||
|| ||
|| Chromosomes/contigs in annotation but not in index : ||
|| NT_166281.1 ||
|| chrY ||
|| NT_187056.1 ||
|| NT_187054.1 ||
|| chr14 ||
|| NT_187060.1 ||
|| NT_187057.1 ||
|| chr8 ||
|| NT_166282.1 ||
|| NT_166307.1 ||
|| chr10 ||
|| NT_187059.1 ||
|| chr11 ||
|| chr2 ||
|| NT_187062.1 ||
|| chrX ||
|| NT_187052.1 ||
|| chr19 ||
|| NT_166438.1 ||
|| NT_166466.1 ||
|| chr12 ||
|| NT_187058.1 ||
|| chrM ||
|| chr7 ||
|| chr18 ||
|| chr3 ||
|| NT_187053.1 ||
|| NT_166291.1 ||
|| chr13 ||
|| chr9 ||
|| chr17 ||
|| chr6 ||
|| NT_162750.1 ||
|| NT_165789.2 ||
|| chr5 ||
|| chr16 ||
|| NT_166463.1 ||
|| chr4 ||
|| NT_166434.1 ||
|| NT_166280.1 ||
|| chr15 ||
|| NT_187064.1 ||
|| ||
|| Global environment is initialised. ||
|| Load the 1-th index block... ||
|| The index block has been loaded. ||
|| Start read mapping in chunk. ||
|| ||
|| Completed successfully. ||
|| ||
\\==================================== ====================================//
//================================ Summary =================================\\
|| ||
|| Total reads : 1000 ||
|| Mapped : 141 (14.1%) ||
|| Uniquely mapped : 129 ||
|| Multi-mapping : 12 ||
|| ||
|| Unmapped : 859 ||
|| ||
|| Indels : 9 ||
|| ||
|| WARNING : Phred offset (64) incorrect? ||
|| ||
|| Running time : 0.4 minutes ||
|| ||
\\============================================================================//
========== _____ _ _ ____ _____ ______ _____
===== / ____| | | | _ \| __ \| ____| /\ | __ \
===== | (___ | | | | |_) | |__) | |__ / \ | | | |
==== \___ \| | | | _ <| _ /| __| / /\ \ | | | |
==== ____) | |__| | |_) | | \ \| |____ / ____ \| |__| |
========== |_____/ \____/|____/|_| \_\______/_/ \_\_____/
Rsubread 2.8.2
//================================= setting ==================================\\
|| ||
|| Function : Read alignment (RNA-Seq) ||
|| Input file : SRR1552445.fastq.gz ||
|| Output file : SRR1552445.fastq.gz.subread.BAM (BAM) ||
|| Index name : chr1_mm10 ||
|| ||
|| ------------------------------------ ||
|| ||
|| Threads : 10 ||
|| Phred offset : 64 ||
|| Min votes : 3 / 10 ||
|| Max mismatches : 3 ||
|| Max indel length : 5 ||
|| Report multi-mapping reads : yes ||
|| Max alignments per multi-mapping read : 1 ||
|| Annotations : inbuilt (mm10) ||
|| ||
\\============================================================================//
//================ Running (01-May-2022 18:27:02, pid=23920) =================\\
|| ||
|| Check the input reads. ||
|| The input file contains base space reads. ||
|| Initialise the memory objects. ||
|| Estimate the mean read length. ||
|| WARNING - The specified Phred score offset (64) seems incorrect. ||
|| ASCII values of the quality scores of read bases included in ||
|| the first 999 reads were found to be within the range of ||
|| [35,74]. ||
|| ||
|| Create the output BAM file. ||
|| Check the index. ||
|| Init the voting space. ||
|| Load the annotation file. ||
|| 222996 annotation records were loaded. ||
|| ||
|| Chromosomes/contigs in annotation but not in index : ||
|| NT_166281.1 ||
|| chrY ||
|| NT_187056.1 ||
|| NT_187054.1 ||
|| chr14 ||
|| NT_187060.1 ||
|| NT_187057.1 ||
|| chr8 ||
|| NT_166282.1 ||
|| NT_166307.1 ||
|| chr10 ||
|| NT_187059.1 ||
|| chr11 ||
|| chr2 ||
|| NT_187062.1 ||
|| chrX ||
|| NT_187052.1 ||
|| chr19 ||
|| NT_166438.1 ||
|| NT_166466.1 ||
|| chr12 ||
|| NT_187058.1 ||
|| chrM ||
|| chr7 ||
|| chr18 ||
|| chr3 ||
|| NT_187053.1 ||
|| NT_166291.1 ||
|| chr13 ||
|| chr9 ||
|| chr17 ||
|| chr6 ||
|| NT_162750.1 ||
|| NT_165789.2 ||
|| chr5 ||
|| chr16 ||
|| NT_166463.1 ||
|| chr4 ||
|| NT_166434.1 ||
|| NT_166280.1 ||
|| chr15 ||
|| NT_187064.1 ||
|| ||
|| Global environment is initialised. ||
|| Load the 1-th index block... ||
|| The index block has been loaded. ||
|| Start read mapping in chunk. ||
|| ||
|| Completed successfully. ||
|| ||
\\==================================== ====================================//
//================================ Summary =================================\\
|| ||
|| Total reads : 1000 ||
|| Mapped : 132 (13.2%) ||
|| Uniquely mapped : 127 ||
|| Multi-mapping : 5 ||
|| ||
|| Unmapped : 868 ||
|| ||
|| Indels : 1 ||
|| ||
|| WARNING : Phred offset (64) incorrect? ||
|| ||
|| Running time : 0.4 minutes ||
|| ||
\\============================================================================//
========== _____ _ _ ____ _____ ______ _____
===== / ____| | | | _ \| __ \| ____| /\ | __ \
===== | (___ | | | | |_) | |__) | |__ / \ | | | |
==== \___ \| | | | _ <| _ /| __| / /\ \ | | | |
==== ____) | |__| | |_) | | \ \| |____ / ____ \| |__| |
========== |_____/ \____/|____/|_| \_\______/_/ \_\_____/
Rsubread 2.8.2
//================================= setting ==================================\\
|| ||
|| Function : Read alignment (RNA-Seq) ||
|| Input file : SRR1552446.fastq.gz ||
|| Output file : SRR1552446.fastq.gz.subread.BAM (BAM) ||
|| Index name : chr1_mm10 ||
|| ||
|| ------------------------------------ ||
|| ||
|| Threads : 10 ||
|| Phred offset : 64 ||
|| Min votes : 3 / 10 ||
|| Max mismatches : 3 ||
|| Max indel length : 5 ||
|| Report multi-mapping reads : yes ||
|| Max alignments per multi-mapping read : 1 ||
|| Annotations : inbuilt (mm10) ||
|| ||
\\============================================================================//
//================ Running (01-May-2022 18:27:26, pid=23920) =================\\
|| ||
|| Check the input reads. ||
|| The input file contains base space reads. ||
|| Initialise the memory objects. ||
|| Estimate the mean read length. ||
|| WARNING - The specified Phred score offset (64) seems incorrect. ||
|| ASCII values of the quality scores of read bases included in ||
|| the first 999 reads were found to be within the range of ||
|| [35,74]. ||
|| ||
|| Create the output BAM file. ||
|| Check the index. ||
|| Init the voting space. ||
|| Load the annotation file. ||
|| 222996 annotation records were loaded. ||
|| ||
|| Chromosomes/contigs in annotation but not in index : ||
|| NT_166281.1 ||
|| chrY ||
|| NT_187056.1 ||
|| NT_187054.1 ||
|| chr14 ||
|| NT_187060.1 ||
|| NT_187057.1 ||
|| chr8 ||
|| NT_166282.1 ||
|| NT_166307.1 ||
|| chr10 ||
|| NT_187059.1 ||
|| chr11 ||
|| chr2 ||
|| NT_187062.1 ||
|| chrX ||
|| NT_187052.1 ||
|| chr19 ||
|| NT_166438.1 ||
|| NT_166466.1 ||
|| chr12 ||
|| NT_187058.1 ||
|| chrM ||
|| chr7 ||
|| chr18 ||
|| chr3 ||
|| NT_187053.1 ||
|| NT_166291.1 ||
|| chr13 ||
|| chr9 ||
|| chr17 ||
|| chr6 ||
|| NT_162750.1 ||
|| NT_165789.2 ||
|| chr5 ||
|| chr16 ||
|| NT_166463.1 ||
|| chr4 ||
|| NT_166434.1 ||
|| NT_166280.1 ||
|| chr15 ||
|| NT_187064.1 ||
|| ||
|| Global environment is initialised. ||
|| Load the 1-th index block... ||
|| The index block has been loaded. ||
|| Start read mapping in chunk. ||
|| ||
|| Completed successfully. ||
|| ||
\\==================================== ====================================//
//================================ Summary =================================\\
|| ||
|| Total reads : 1000 ||
|| Mapped : 119 (11.9%) ||
|| Uniquely mapped : 111 ||
|| Multi-mapping : 8 ||
|| ||
|| Unmapped : 881 ||
|| ||
|| Indels : 7 ||
|| ||
|| WARNING : Phred offset (64) incorrect? ||
|| ||
|| Running time : 0.4 minutes ||
|| ||
\\============================================================================//
========== _____ _ _ ____ _____ ______ _____
===== / ____| | | | _ \| __ \| ____| /\ | __ \
===== | (___ | | | | |_) | |__) | |__ / \ | | | |
==== \___ \| | | | _ <| _ /| __| / /\ \ | | | |
==== ____) | |__| | |_) | | \ \| |____ / ____ \| |__| |
========== |_____/ \____/|____/|_| \_\______/_/ \_\_____/
Rsubread 2.8.2
//================================= setting ==================================\\
|| ||
|| Function : Read alignment (RNA-Seq) ||
|| Input file : SRR1552447.fastq.gz ||
|| Output file : SRR1552447.fastq.gz.subread.BAM (BAM) ||
|| Index name : chr1_mm10 ||
|| ||
|| ------------------------------------ ||
|| ||
|| Threads : 10 ||
|| Phred offset : 64 ||
|| Min votes : 3 / 10 ||
|| Max mismatches : 3 ||
|| Max indel length : 5 ||
|| Report multi-mapping reads : yes ||
|| Max alignments per multi-mapping read : 1 ||
|| Annotations : inbuilt (mm10) ||
|| ||
\\============================================================================//
//================ Running (01-May-2022 18:27:49, pid=23920) =================\\
|| ||
|| Check the input reads. ||
|| The input file contains base space reads. ||
|| Initialise the memory objects. ||
|| Estimate the mean read length. ||
|| WARNING - The specified Phred score offset (64) seems incorrect. ||
|| ASCII values of the quality scores of read bases included in ||
|| the first 999 reads were found to be within the range of ||
|| [35,74]. ||
|| ||
|| Create the output BAM file. ||
|| Check the index. ||
|| Init the voting space. ||
|| Load the annotation file. ||
|| 222996 annotation records were loaded. ||
|| ||
|| Chromosomes/contigs in annotation but not in index : ||
|| NT_166281.1 ||
|| chrY ||
|| NT_187056.1 ||
|| NT_187054.1 ||
|| chr14 ||
|| NT_187060.1 ||
|| NT_187057.1 ||
|| chr8 ||
|| NT_166282.1 ||
|| NT_166307.1 ||
|| chr10 ||
|| NT_187059.1 ||
|| chr11 ||
|| chr2 ||
|| NT_187062.1 ||
|| chrX ||
|| NT_187052.1 ||
|| chr19 ||
|| NT_166438.1 ||
|| NT_166466.1 ||
|| chr12 ||
|| NT_187058.1 ||
|| chrM ||
|| chr7 ||
|| chr18 ||
|| chr3 ||
|| NT_187053.1 ||
|| NT_166291.1 ||
|| chr13 ||
|| chr9 ||
|| chr17 ||
|| chr6 ||
|| NT_162750.1 ||
|| NT_165789.2 ||
|| chr5 ||
|| chr16 ||
|| NT_166463.1 ||
|| chr4 ||
|| NT_166434.1 ||
|| NT_166280.1 ||
|| chr15 ||
|| NT_187064.1 ||
|| ||
|| Global environment is initialised. ||
|| Load the 1-th index block... ||
|| The index block has been loaded. ||
|| Start read mapping in chunk. ||
|| ||
|| Completed successfully. ||
|| ||
\\==================================== ====================================//
//================================ Summary =================================\\
|| ||
|| Total reads : 1000 ||
|| Mapped : 112 (11.2%) ||
|| Uniquely mapped : 104 ||
|| Multi-mapping : 8 ||
|| ||
|| Unmapped : 888 ||
|| ||
|| Indels : 3 ||
|| ||
|| WARNING : Phred offset (64) incorrect? ||
|| ||
|| Running time : 0.4 minutes ||
|| ||
\\============================================================================//
========== _____ _ _ ____ _____ ______ _____
===== / ____| | | | _ \| __ \| ____| /\ | __ \
===== | (___ | | | | |_) | |__) | |__ / \ | | | |
==== \___ \| | | | _ <| _ /| __| / /\ \ | | | |
==== ____) | |__| | |_) | | \ \| |____ / ____ \| |__| |
========== |_____/ \____/|____/|_| \_\______/_/ \_\_____/
Rsubread 2.8.2
//================================= setting ==================================\\
|| ||
|| Function : Read alignment (RNA-Seq) ||
|| Input file : SRR1552448.fastq.gz ||
|| Output file : SRR1552448.fastq.gz.subread.BAM (BAM) ||
|| Index name : chr1_mm10 ||
|| ||
|| ------------------------------------ ||
|| ||
|| Threads : 10 ||
|| Phred offset : 64 ||
|| Min votes : 3 / 10 ||
|| Max mismatches : 3 ||
|| Max indel length : 5 ||
|| Report multi-mapping reads : yes ||
|| Max alignments per multi-mapping read : 1 ||
|| Annotations : inbuilt (mm10) ||
|| ||
\\============================================================================//
//================ Running (01-May-2022 18:28:13, pid=23920) =================\\
|| ||
|| Check the input reads. ||
|| The input file contains base space reads. ||
|| Initialise the memory objects. ||
|| Estimate the mean read length. ||
|| WARNING - The specified Phred score offset (64) seems incorrect. ||
|| ASCII values of the quality scores of read bases included in ||
|| the first 999 reads were found to be within the range of ||
|| [35,74]. ||
|| ||
|| Create the output BAM file. ||
|| Check the index. ||
|| Init the voting space. ||
|| Load the annotation file. ||
|| 222996 annotation records were loaded. ||
|| ||
|| Chromosomes/contigs in annotation but not in index : ||
|| NT_166281.1 ||
|| chrY ||
|| NT_187056.1 ||
|| NT_187054.1 ||
|| chr14 ||
|| NT_187060.1 ||
|| NT_187057.1 ||
|| chr8 ||
|| NT_166282.1 ||
|| NT_166307.1 ||
|| chr10 ||
|| NT_187059.1 ||
|| chr11 ||
|| chr2 ||
|| NT_187062.1 ||
|| chrX ||
|| NT_187052.1 ||
|| chr19 ||
|| NT_166438.1 ||
|| NT_166466.1 ||
|| chr12 ||
|| NT_187058.1 ||
|| chrM ||
|| chr7 ||
|| chr18 ||
|| chr3 ||
|| NT_187053.1 ||
|| NT_166291.1 ||
|| chr13 ||
|| chr9 ||
|| chr17 ||
|| chr6 ||
|| NT_162750.1 ||
|| NT_165789.2 ||
|| chr5 ||
|| chr16 ||
|| NT_166463.1 ||
|| chr4 ||
|| NT_166434.1 ||
|| NT_166280.1 ||
|| chr15 ||
|| NT_187064.1 ||
|| ||
|| Global environment is initialised. ||
|| Load the 1-th index block... ||
|| The index block has been loaded. ||
|| Start read mapping in chunk. ||
|| ||
|| Completed successfully. ||
|| ||
\\==================================== ====================================//
//================================ Summary =================================\\
|| ||
|| Total reads : 1000 ||
|| Mapped : 53 (5.3%) ||
|| Uniquely mapped : 51 ||
|| Multi-mapping : 2 ||
|| ||
|| Unmapped : 947 ||
|| ||
|| Indels : 1 ||
|| ||
|| WARNING : Phred offset (64) incorrect? ||
|| ||
|| Running time : 0.4 minutes ||
|| ||
\\============================================================================//
========== _____ _ _ ____ _____ ______ _____
===== / ____| | | | _ \| __ \| ____| /\ | __ \
===== | (___ | | | | |_) | |__) | |__ / \ | | | |
==== \___ \| | | | _ <| _ /| __| / /\ \ | | | |
==== ____) | |__| | |_) | | \ \| |____ / ____ \| |__| |
========== |_____/ \____/|____/|_| \_\______/_/ \_\_____/
Rsubread 2.8.2
//================================= setting ==================================\\
|| ||
|| Function : Read alignment (RNA-Seq) ||
|| Input file : SRR1552449.fastq.gz ||
|| Output file : SRR1552449.fastq.gz.subread.BAM (BAM) ||
|| Index name : chr1_mm10 ||
|| ||
|| ------------------------------------ ||
|| ||
|| Threads : 10 ||
|| Phred offset : 64 ||
|| Min votes : 3 / 10 ||
|| Max mismatches : 3 ||
|| Max indel length : 5 ||
|| Report multi-mapping reads : yes ||
|| Max alignments per multi-mapping read : 1 ||
|| Annotations : inbuilt (mm10) ||
|| ||
\\============================================================================//
//================ Running (01-May-2022 18:28:36, pid=23920) =================\\
|| ||
|| Check the input reads. ||
|| The input file contains base space reads. ||
|| Initialise the memory objects. ||
|| Estimate the mean read length. ||
|| WARNING - The specified Phred score offset (64) seems incorrect. ||
|| ASCII values of the quality scores of read bases included in ||
|| the first 999 reads were found to be within the range of ||
|| [35,74]. ||
|| ||
|| Create the output BAM file. ||
|| Check the index. ||
|| Init the voting space. ||
|| Load the annotation file. ||
|| 222996 annotation records were loaded. ||
|| ||
|| Chromosomes/contigs in annotation but not in index : ||
|| NT_166281.1 ||
|| chrY ||
|| NT_187056.1 ||
|| NT_187054.1 ||
|| chr14 ||
|| NT_187060.1 ||
|| NT_187057.1 ||
|| chr8 ||
|| NT_166282.1 ||
|| NT_166307.1 ||
|| chr10 ||
|| NT_187059.1 ||
|| chr11 ||
|| chr2 ||
|| NT_187062.1 ||
|| chrX ||
|| NT_187052.1 ||
|| chr19 ||
|| NT_166438.1 ||
|| NT_166466.1 ||
|| chr12 ||
|| NT_187058.1 ||
|| chrM ||
|| chr7 ||
|| chr18 ||
|| chr3 ||
|| NT_187053.1 ||
|| NT_166291.1 ||
|| chr13 ||
|| chr9 ||
|| chr17 ||
|| chr6 ||
|| NT_162750.1 ||
|| NT_165789.2 ||
|| chr5 ||
|| chr16 ||
|| NT_166463.1 ||
|| chr4 ||
|| NT_166434.1 ||
|| NT_166280.1 ||
|| chr15 ||
|| NT_187064.1 ||
|| ||
|| Global environment is initialised. ||
|| Load the 1-th index block... ||
|| The index block has been loaded. ||
|| Start read mapping in chunk. ||
|| ||
|| Completed successfully. ||
|| ||
\\==================================== ====================================//
//================================ Summary =================================\\
|| ||
|| Total reads : 1000 ||
|| Mapped : 75 (7.5%) ||
|| Uniquely mapped : 66 ||
|| Multi-mapping : 9 ||
|| ||
|| Unmapped : 925 ||
|| ||
|| Indels : 2 ||
|| ||
|| WARNING : Phred offset (64) incorrect? ||
|| ||
|| Running time : 0.4 minutes ||
|| ||
\\============================================================================//
========== _____ _ _ ____ _____ ______ _____
===== / ____| | | | _ \| __ \| ____| /\ | __ \
===== | (___ | | | | |_) | |__) | |__ / \ | | | |
==== \___ \| | | | _ <| _ /| __| / /\ \ | | | |
==== ____) | |__| | |_) | | \ \| |____ / ____ \| |__| |
========== |_____/ \____/|____/|_| \_\______/_/ \_\_____/
Rsubread 2.8.2
//================================= setting ==================================\\
|| ||
|| Function : Read alignment (RNA-Seq) ||
|| Input file : SRR1552450.fastq.gz ||
|| Output file : SRR1552450.fastq.gz.subread.BAM (BAM) ||
|| Index name : chr1_mm10 ||
|| ||
|| ------------------------------------ ||
|| ||
|| Threads : 10 ||
|| Phred offset : 64 ||
|| Min votes : 3 / 10 ||
|| Max mismatches : 3 ||
|| Max indel length : 5 ||
|| Report multi-mapping reads : yes ||
|| Max alignments per multi-mapping read : 1 ||
|| Annotations : inbuilt (mm10) ||
|| ||
\\============================================================================//
//================ Running (01-May-2022 18:29:00, pid=23920) =================\\
|| ||
|| Check the input reads. ||
|| The input file contains base space reads. ||
|| Initialise the memory objects. ||
|| Estimate the mean read length. ||
|| WARNING - The specified Phred score offset (64) seems incorrect. ||
|| ASCII values of the quality scores of read bases included in ||
|| the first 999 reads were found to be within the range of ||
|| [35,74]. ||
|| ||
|| Create the output BAM file. ||
|| Check the index. ||
|| Init the voting space. ||
|| Load the annotation file. ||
|| 222996 annotation records were loaded. ||
|| ||
|| Chromosomes/contigs in annotation but not in index : ||
|| NT_166281.1 ||
|| chrY ||
|| NT_187056.1 ||
|| NT_187054.1 ||
|| chr14 ||
|| NT_187060.1 ||
|| NT_187057.1 ||
|| chr8 ||
|| NT_166282.1 ||
|| NT_166307.1 ||
|| chr10 ||
|| NT_187059.1 ||
|| chr11 ||
|| chr2 ||
|| NT_187062.1 ||
|| chrX ||
|| NT_187052.1 ||
|| chr19 ||
|| NT_166438.1 ||
|| NT_166466.1 ||
|| chr12 ||
|| NT_187058.1 ||
|| chrM ||
|| chr7 ||
|| chr18 ||
|| chr3 ||
|| NT_187053.1 ||
|| NT_166291.1 ||
|| chr13 ||
|| chr9 ||
|| chr17 ||
|| chr6 ||
|| NT_162750.1 ||
|| NT_165789.2 ||
|| chr5 ||
|| chr16 ||
|| NT_166463.1 ||
|| chr4 ||
|| NT_166434.1 ||
|| NT_166280.1 ||
|| chr15 ||
|| NT_187064.1 ||
|| ||
|| Global environment is initialised. ||
|| Load the 1-th index block... ||
|| The index block has been loaded. ||
|| Start read mapping in chunk. ||
|| ||
|| Completed successfully. ||
|| ||
\\==================================== ====================================//
//================================ Summary =================================\\
|| ||
|| Total reads : 1000 ||
|| Mapped : 124 (12.4%) ||
|| Uniquely mapped : 119 ||
|| Multi-mapping : 5 ||
|| ||
|| Unmapped : 876 ||
|| ||
|| Indels : 4 ||
|| ||
|| WARNING : Phred offset (64) incorrect? ||
|| ||
|| Running time : 0.4 minutes ||
|| ||
\\============================================================================//
========== _____ _ _ ____ _____ ______ _____
===== / ____| | | | _ \| __ \| ____| /\ | __ \
===== | (___ | | | | |_) | |__) | |__ / \ | | | |
==== \___ \| | | | _ <| _ /| __| / /\ \ | | | |
==== ____) | |__| | |_) | | \ \| |____ / ____ \| |__| |
========== |_____/ \____/|____/|_| \_\______/_/ \_\_____/
Rsubread 2.8.2
//================================= setting ==================================\\
|| ||
|| Function : Read alignment (RNA-Seq) ||
|| Input file : SRR1552451.fastq.gz ||
|| Output file : SRR1552451.fastq.gz.subread.BAM (BAM) ||
|| Index name : chr1_mm10 ||
|| ||
|| ------------------------------------ ||
|| ||
|| Threads : 10 ||
|| Phred offset : 64 ||
|| Min votes : 3 / 10 ||
|| Max mismatches : 3 ||
|| Max indel length : 5 ||
|| Report multi-mapping reads : yes ||
|| Max alignments per multi-mapping read : 1 ||
|| Annotations : inbuilt (mm10) ||
|| ||
\\============================================================================//
//================ Running (01-May-2022 18:29:23, pid=23920) =================\\
|| ||
|| Check the input reads. ||
|| The input file contains base space reads. ||
|| Initialise the memory objects. ||
|| Estimate the mean read length. ||
|| WARNING - The specified Phred score offset (64) seems incorrect. ||
|| ASCII values of the quality scores of read bases included in ||
|| the first 999 reads were found to be within the range of ||
|| [35,74]. ||
|| ||
|| Create the output BAM file. ||
|| Check the index. ||
|| Init the voting space. ||
|| Load the annotation file. ||
|| 222996 annotation records were loaded. ||
|| ||
|| Chromosomes/contigs in annotation but not in index : ||
|| NT_166281.1 ||
|| chrY ||
|| NT_187056.1 ||
|| NT_187054.1 ||
|| chr14 ||
|| NT_187060.1 ||
|| NT_187057.1 ||
|| chr8 ||
|| NT_166282.1 ||
|| NT_166307.1 ||
|| chr10 ||
|| NT_187059.1 ||
|| chr11 ||
|| chr2 ||
|| NT_187062.1 ||
|| chrX ||
|| NT_187052.1 ||
|| chr19 ||
|| NT_166438.1 ||
|| NT_166466.1 ||
|| chr12 ||
|| NT_187058.1 ||
|| chrM ||
|| chr7 ||
|| chr18 ||
|| chr3 ||
|| NT_187053.1 ||
|| NT_166291.1 ||
|| chr13 ||
|| chr9 ||
|| chr17 ||
|| chr6 ||
|| NT_162750.1 ||
|| NT_165789.2 ||
|| chr5 ||
|| chr16 ||
|| NT_166463.1 ||
|| chr4 ||
|| NT_166434.1 ||
|| NT_166280.1 ||
|| chr15 ||
|| NT_187064.1 ||
|| ||
|| Global environment is initialised. ||
|| Load the 1-th index block... ||
|| The index block has been loaded. ||
|| Start read mapping in chunk. ||
|| ||
|| Completed successfully. ||
|| ||
\\==================================== ====================================//
//================================ Summary =================================\\
|| ||
|| Total reads : 1000 ||
|| Mapped : 158 (15.8%) ||
|| Uniquely mapped : 143 ||
|| Multi-mapping : 15 ||
|| ||
|| Unmapped : 842 ||
|| ||
|| Indels : 9 ||
|| ||
|| WARNING : Phred offset (64) incorrect? ||
|| ||
|| Running time : 0.4 minutes ||
|| ||
\\============================================================================//
========== _____ _ _ ____ _____ ______ _____
===== / ____| | | | _ \| __ \| ____| /\ | __ \
===== | (___ | | | | |_) | |__) | |__ / \ | | | |
==== \___ \| | | | _ <| _ /| __| / /\ \ | | | |
==== ____) | |__| | |_) | | \ \| |____ / ____ \| |__| |
========== |_____/ \____/|____/|_| \_\______/_/ \_\_____/
Rsubread 2.8.2
//================================= setting ==================================\\
|| ||
|| Function : Read alignment (RNA-Seq) ||
|| Input file : SRR1552452.fastq.gz ||
|| Output file : SRR1552452.fastq.gz.subread.BAM (BAM) ||
|| Index name : chr1_mm10 ||
|| ||
|| ------------------------------------ ||
|| ||
|| Threads : 10 ||
|| Phred offset : 64 ||
|| Min votes : 3 / 10 ||
|| Max mismatches : 3 ||
|| Max indel length : 5 ||
|| Report multi-mapping reads : yes ||
|| Max alignments per multi-mapping read : 1 ||
|| Annotations : inbuilt (mm10) ||
|| ||
\\============================================================================//
//================ Running (01-May-2022 18:29:47, pid=23920) =================\\
|| ||
|| Check the input reads. ||
|| The input file contains base space reads. ||
|| Initialise the memory objects. ||
|| Estimate the mean read length. ||
|| WARNING - The specified Phred score offset (64) seems incorrect. ||
|| ASCII values of the quality scores of read bases included in ||
|| the first 999 reads were found to be within the range of ||
|| [35,74]. ||
|| ||
|| Create the output BAM file. ||
|| Check the index. ||
|| Init the voting space. ||
|| Load the annotation file. ||
|| 222996 annotation records were loaded. ||
|| ||
|| Chromosomes/contigs in annotation but not in index : ||
|| NT_166281.1 ||
|| chrY ||
|| NT_187056.1 ||
|| NT_187054.1 ||
|| chr14 ||
|| NT_187060.1 ||
|| NT_187057.1 ||
|| chr8 ||
|| NT_166282.1 ||
|| NT_166307.1 ||
|| chr10 ||
|| NT_187059.1 ||
|| chr11 ||
|| chr2 ||
|| NT_187062.1 ||
|| chrX ||
|| NT_187052.1 ||
|| chr19 ||
|| NT_166438.1 ||
|| NT_166466.1 ||
|| chr12 ||
|| NT_187058.1 ||
|| chrM ||
|| chr7 ||
|| chr18 ||
|| chr3 ||
|| NT_187053.1 ||
|| NT_166291.1 ||
|| chr13 ||
|| chr9 ||
|| chr17 ||
|| chr6 ||
|| NT_162750.1 ||
|| NT_165789.2 ||
|| chr5 ||
|| chr16 ||
|| NT_166463.1 ||
|| chr4 ||
|| NT_166434.1 ||
|| NT_166280.1 ||
|| chr15 ||
|| NT_187064.1 ||
|| ||
|| Global environment is initialised. ||
|| Load the 1-th index block... ||
|| The index block has been loaded. ||
|| Start read mapping in chunk. ||
|| ||
|| Completed successfully. ||
|| ||
\\==================================== ====================================//
//================================ Summary =================================\\
|| ||
|| Total reads : 1000 ||
|| Mapped : 142 (14.2%) ||
|| Uniquely mapped : 133 ||
|| Multi-mapping : 9 ||
|| ||
|| Unmapped : 858 ||
|| ||
|| Indels : 6 ||
|| ||
|| WARNING : Phred offset (64) incorrect? ||
|| ||
|| Running time : 0.4 minutes ||
|| ||
\\============================================================================//
========== _____ _ _ ____ _____ ______ _____
===== / ____| | | | _ \| __ \| ____| /\ | __ \
===== | (___ | | | | |_) | |__) | |__ / \ | | | |
==== \___ \| | | | _ <| _ /| __| / /\ \ | | | |
==== ____) | |__| | |_) | | \ \| |____ / ____ \| |__| |
========== |_____/ \____/|____/|_| \_\______/_/ \_\_____/
Rsubread 2.8.2
//================================= setting ==================================\\
|| ||
|| Function : Read alignment (RNA-Seq) ||
|| Input file : SRR1552453.fastq.gz ||
|| Output file : SRR1552453.fastq.gz.subread.BAM (BAM) ||
|| Index name : chr1_mm10 ||
|| ||
|| ------------------------------------ ||
|| ||
|| Threads : 10 ||
|| Phred offset : 64 ||
|| Min votes : 3 / 10 ||
|| Max mismatches : 3 ||
|| Max indel length : 5 ||
|| Report multi-mapping reads : yes ||
|| Max alignments per multi-mapping read : 1 ||
|| Annotations : inbuilt (mm10) ||
|| ||
\\============================================================================//
//================ Running (01-May-2022 18:30:11, pid=23920) =================\\
|| ||
|| Check the input reads. ||
|| The input file contains base space reads. ||
|| Initialise the memory objects. ||
|| Estimate the mean read length. ||
|| WARNING - The specified Phred score offset (64) seems incorrect. ||
|| ASCII values of the quality scores of read bases included in ||
|| the first 999 reads were found to be within the range of ||
|| [35,74]. ||
|| ||
|| Create the output BAM file. ||
|| Check the index. ||
|| Init the voting space. ||
|| Load the annotation file. ||
|| 222996 annotation records were loaded. ||
|| ||
|| Chromosomes/contigs in annotation but not in index : ||
|| NT_166281.1 ||
|| chrY ||
|| NT_187056.1 ||
|| NT_187054.1 ||
|| chr14 ||
|| NT_187060.1 ||
|| NT_187057.1 ||
|| chr8 ||
|| NT_166282.1 ||
|| NT_166307.1 ||
|| chr10 ||
|| NT_187059.1 ||
|| chr11 ||
|| chr2 ||
|| NT_187062.1 ||
|| chrX ||
|| NT_187052.1 ||
|| chr19 ||
|| NT_166438.1 ||
|| NT_166466.1 ||
|| chr12 ||
|| NT_187058.1 ||
|| chrM ||
|| chr7 ||
|| chr18 ||
|| chr3 ||
|| NT_187053.1 ||
|| NT_166291.1 ||
|| chr13 ||
|| chr9 ||
|| chr17 ||
|| chr6 ||
|| NT_162750.1 ||
|| NT_165789.2 ||
|| chr5 ||
|| chr16 ||
|| NT_166463.1 ||
|| chr4 ||
|| NT_166434.1 ||
|| NT_166280.1 ||
|| chr15 ||
|| NT_187064.1 ||
|| ||
|| Global environment is initialised. ||
|| Load the 1-th index block... ||
|| The index block has been loaded. ||
|| Start read mapping in chunk. ||
|| ||
|| Completed successfully. ||
|| ||
\\==================================== ====================================//
//================================ Summary =================================\\
|| ||
|| Total reads : 1000 ||
|| Mapped : 147 (14.7%) ||
|| Uniquely mapped : 137 ||
|| Multi-mapping : 10 ||
|| ||
|| Unmapped : 853 ||
|| ||
|| Indels : 5 ||
|| ||
|| WARNING : Phred offset (64) incorrect? ||
|| ||
|| Running time : 0.4 minutes ||
|| ||
\\============================================================================//
========== _____ _ _ ____ _____ ______ _____
===== / ____| | | | _ \| __ \| ____| /\ | __ \
===== | (___ | | | | |_) | |__) | |__ / \ | | | |
==== \___ \| | | | _ <| _ /| __| / /\ \ | | | |
==== ____) | |__| | |_) | | \ \| |____ / ____ \| |__| |
========== |_____/ \____/|____/|_| \_\______/_/ \_\_____/
Rsubread 2.8.2
//================================= setting ==================================\\
|| ||
|| Function : Read alignment (RNA-Seq) ||
|| Input file : SRR1552454.fastq.gz ||
|| Output file : SRR1552454.fastq.gz.subread.BAM (BAM) ||
|| Index name : chr1_mm10 ||
|| ||
|| ------------------------------------ ||
|| ||
|| Threads : 10 ||
|| Phred offset : 64 ||
|| Min votes : 3 / 10 ||
|| Max mismatches : 3 ||
|| Max indel length : 5 ||
|| Report multi-mapping reads : yes ||
|| Max alignments per multi-mapping read : 1 ||
|| Annotations : inbuilt (mm10) ||
|| ||
\\============================================================================//
//================ Running (01-May-2022 18:30:34, pid=23920) =================\\
|| ||
|| Check the input reads. ||
|| The input file contains base space reads. ||
|| Initialise the memory objects. ||
|| Estimate the mean read length. ||
|| WARNING - The specified Phred score offset (64) seems incorrect. ||
|| ASCII values of the quality scores of read bases included in ||
|| the first 999 reads were found to be within the range of ||
|| [35,74]. ||
|| ||
|| Create the output BAM file. ||
|| Check the index. ||
|| Init the voting space. ||
|| Load the annotation file. ||
|| 222996 annotation records were loaded. ||
|| ||
|| Chromosomes/contigs in annotation but not in index : ||
|| NT_166281.1 ||
|| chrY ||
|| NT_187056.1 ||
|| NT_187054.1 ||
|| chr14 ||
|| NT_187060.1 ||
|| NT_187057.1 ||
|| chr8 ||
|| NT_166282.1 ||
|| NT_166307.1 ||
|| chr10 ||
|| NT_187059.1 ||
|| chr11 ||
|| chr2 ||
|| NT_187062.1 ||
|| chrX ||
|| NT_187052.1 ||
|| chr19 ||
|| NT_166438.1 ||
|| NT_166466.1 ||
|| chr12 ||
|| NT_187058.1 ||
|| chrM ||
|| chr7 ||
|| chr18 ||
|| chr3 ||
|| NT_187053.1 ||
|| NT_166291.1 ||
|| chr13 ||
|| chr9 ||
|| chr17 ||
|| chr6 ||
|| NT_162750.1 ||
|| NT_165789.2 ||
|| chr5 ||
|| chr16 ||
|| NT_166463.1 ||
|| chr4 ||
|| NT_166434.1 ||
|| NT_166280.1 ||
|| chr15 ||
|| NT_187064.1 ||
|| ||
|| Global environment is initialised. ||
|| Load the 1-th index block... ||
|| The index block has been loaded. ||
|| Start read mapping in chunk. ||
|| ||
|| Completed successfully. ||
|| ||
\\==================================== ====================================//
//================================ Summary =================================\\
|| ||
|| Total reads : 1000 ||
|| Mapped : 130 (13.0%) ||
|| Uniquely mapped : 126 ||
|| Multi-mapping : 4 ||
|| ||
|| Unmapped : 870 ||
|| ||
|| Indels : 2 ||
|| ||
|| WARNING : Phred offset (64) incorrect? ||
|| ||
|| Running time : 0.4 minutes ||
|| ||
\\============================================================================//
========== _____ _ _ ____ _____ ______ _____
===== / ____| | | | _ \| __ \| ____| /\ | __ \
===== | (___ | | | | |_) | |__) | |__ / \ | | | |
==== \___ \| | | | _ <| _ /| __| / /\ \ | | | |
==== ____) | |__| | |_) | | \ \| |____ / ____ \| |__| |
========== |_____/ \____/|____/|_| \_\______/_/ \_\_____/
Rsubread 2.8.2
//================================= setting ==================================\\
|| ||
|| Function : Read alignment (RNA-Seq) ||
|| Input file : SRR1552455.fastq.gz ||
|| Output file : SRR1552455.fastq.gz.subread.BAM (BAM) ||
|| Index name : chr1_mm10 ||
|| ||
|| ------------------------------------ ||
|| ||
|| Threads : 10 ||
|| Phred offset : 64 ||
|| Min votes : 3 / 10 ||
|| Max mismatches : 3 ||
|| Max indel length : 5 ||
|| Report multi-mapping reads : yes ||
|| Max alignments per multi-mapping read : 1 ||
|| Annotations : inbuilt (mm10) ||
|| ||
\\============================================================================//
//================ Running (01-May-2022 18:30:58, pid=23920) =================\\
|| ||
|| Check the input reads. ||
|| The input file contains base space reads. ||
|| Initialise the memory objects. ||
|| Estimate the mean read length. ||
|| WARNING - The specified Phred score offset (64) seems incorrect. ||
|| ASCII values of the quality scores of read bases included in ||
|| the first 999 reads were found to be within the range of ||
|| [35,74]. ||
|| ||
|| Create the output BAM file. ||
|| Check the index. ||
|| Init the voting space. ||
|| Load the annotation file. ||
|| 222996 annotation records were loaded. ||
|| ||
|| Chromosomes/contigs in annotation but not in index : ||
|| NT_166281.1 ||
|| chrY ||
|| NT_187056.1 ||
|| NT_187054.1 ||
|| chr14 ||
|| NT_187060.1 ||
|| NT_187057.1 ||
|| chr8 ||
|| NT_166282.1 ||
|| NT_166307.1 ||
|| chr10 ||
|| NT_187059.1 ||
|| chr11 ||
|| chr2 ||
|| NT_187062.1 ||
|| chrX ||
|| NT_187052.1 ||
|| chr19 ||
|| NT_166438.1 ||
|| NT_166466.1 ||
|| chr12 ||
|| NT_187058.1 ||
|| chrM ||
|| chr7 ||
|| chr18 ||
|| chr3 ||
|| NT_187053.1 ||
|| NT_166291.1 ||
|| chr13 ||
|| chr9 ||
|| chr17 ||
|| chr6 ||
|| NT_162750.1 ||
|| NT_165789.2 ||
|| chr5 ||
|| chr16 ||
|| NT_166463.1 ||
|| chr4 ||
|| NT_166434.1 ||
|| NT_166280.1 ||
|| chr15 ||
|| NT_187064.1 ||
|| ||
|| Global environment is initialised. ||
|| Load the 1-th index block... ||
|| The index block has been loaded. ||
|| Start read mapping in chunk. ||
|| ||
|| Completed successfully. ||
|| ||
\\==================================== ====================================//
//================================ Summary =================================\\
|| ||
|| Total reads : 1000 ||
|| Mapped : 117 (11.7%) ||
|| Uniquely mapped : 112 ||
|| Multi-mapping : 5 ||
|| ||
|| Unmapped : 883 ||
|| ||
|| Indels : 6 ||
|| ||
|| WARNING : Phred offset (64) incorrect? ||
|| ||
|| Running time : 0.4 minutes ||
|| ||
\\============================================================================// SRR1552444.fastq.gz.subread.BAM
Total_reads 1000
Mapped_reads 141
Uniquely_mapped_reads 129
Multi_mapping_reads 12
Unmapped_reads 859
Indels 9
SRR1552445.fastq.gz.subread.BAM
Total_reads 1000
Mapped_reads 132
Uniquely_mapped_reads 127
Multi_mapping_reads 5
Unmapped_reads 868
Indels 1
SRR1552446.fastq.gz.subread.BAM
Total_reads 1000
Mapped_reads 119
Uniquely_mapped_reads 111
Multi_mapping_reads 8
Unmapped_reads 881
Indels 7
SRR1552447.fastq.gz.subread.BAM
Total_reads 1000
Mapped_reads 112
Uniquely_mapped_reads 104
Multi_mapping_reads 8
Unmapped_reads 888
Indels 3
SRR1552448.fastq.gz.subread.BAM
Total_reads 1000
Mapped_reads 53
Uniquely_mapped_reads 51
Multi_mapping_reads 2
Unmapped_reads 947
Indels 1
SRR1552449.fastq.gz.subread.BAM
Total_reads 1000
Mapped_reads 75
Uniquely_mapped_reads 66
Multi_mapping_reads 9
Unmapped_reads 925
Indels 2
SRR1552450.fastq.gz.subread.BAM
Total_reads 1000
Mapped_reads 124
Uniquely_mapped_reads 119
Multi_mapping_reads 5
Unmapped_reads 876
Indels 4
SRR1552451.fastq.gz.subread.BAM
Total_reads 1000
Mapped_reads 158
Uniquely_mapped_reads 143
Multi_mapping_reads 15
Unmapped_reads 842
Indels 9
SRR1552452.fastq.gz.subread.BAM
Total_reads 1000
Mapped_reads 142
Uniquely_mapped_reads 133
Multi_mapping_reads 9
Unmapped_reads 858
Indels 6
SRR1552453.fastq.gz.subread.BAM
Total_reads 1000
Mapped_reads 147
Uniquely_mapped_reads 137
Multi_mapping_reads 10
Unmapped_reads 853
Indels 5
SRR1552454.fastq.gz.subread.BAM
Total_reads 1000
Mapped_reads 130
Uniquely_mapped_reads 126
Multi_mapping_reads 4
Unmapped_reads 870
Indels 2
SRR1552455.fastq.gz.subread.BAM
Total_reads 1000
Mapped_reads 117
Uniquely_mapped_reads 112
Multi_mapping_reads 5
Unmapped_reads 883
Indels 6If we have paired end data, we would need to specify the second read file/s using the readfile2 argument. The mouse data for this experiment are 100bp single end reads.(this might take very long time to complete)
We can specify the output files, or we can let Rsubread choose the output file names for us. The default output file name is the filename with “.subread.BAM” added at the end.
8.4.
Get a summary of the proportion of reads that mapped to the reference
genome using the propmapped function:
bam.files <- list.files(path = "C:/Users/manso/OneDrive - University of West London/MSc Bioinformatics - UWL/6.BGA - Bioinformatics and Genome Analysis/week 2 - NGS data QC and alignment in R/practical/MouseMamRNA-Seq", pattern = ".BAM$", full.names = TRUE)
bam.files
[1] "C:/Users/manso/OneDrive - University of West London/MSc Bioinformatics - UWL/6.BGA - Bioinformatics and Genome Analysis/week 2 - NGS data QC and alignment in R/practical/MouseMamRNA-Seq/SRR1552444.fastq.gz.subread.BAM"
[2] "C:/Users/manso/OneDrive - University of West London/MSc Bioinformatics - UWL/6.BGA - Bioinformatics and Genome Analysis/week 2 - NGS data QC and alignment in R/practical/MouseMamRNA-Seq/SRR1552445.fastq.gz.subread.BAM"
[3] "C:/Users/manso/OneDrive - University of West London/MSc Bioinformatics - UWL/6.BGA - Bioinformatics and Genome Analysis/week 2 - NGS data QC and alignment in R/practical/MouseMamRNA-Seq/SRR1552446.fastq.gz.subread.BAM"
[4] "C:/Users/manso/OneDrive - University of West London/MSc Bioinformatics - UWL/6.BGA - Bioinformatics and Genome Analysis/week 2 - NGS data QC and alignment in R/practical/MouseMamRNA-Seq/SRR1552447.fastq.gz.subread.BAM"
[5] "C:/Users/manso/OneDrive - University of West London/MSc Bioinformatics - UWL/6.BGA - Bioinformatics and Genome Analysis/week 2 - NGS data QC and alignment in R/practical/MouseMamRNA-Seq/SRR1552448.fastq.gz.subread.BAM"
[6] "C:/Users/manso/OneDrive - University of West London/MSc Bioinformatics - UWL/6.BGA - Bioinformatics and Genome Analysis/week 2 - NGS data QC and alignment in R/practical/MouseMamRNA-Seq/SRR1552449.fastq.gz.subread.BAM"
[7] "C:/Users/manso/OneDrive - University of West London/MSc Bioinformatics - UWL/6.BGA - Bioinformatics and Genome Analysis/week 2 - NGS data QC and alignment in R/practical/MouseMamRNA-Seq/SRR1552450.fastq.gz.subread.BAM"
[8] "C:/Users/manso/OneDrive - University of West London/MSc Bioinformatics - UWL/6.BGA - Bioinformatics and Genome Analysis/week 2 - NGS data QC and alignment in R/practical/MouseMamRNA-Seq/SRR1552451.fastq.gz.subread.BAM"
[9] "C:/Users/manso/OneDrive - University of West London/MSc Bioinformatics - UWL/6.BGA - Bioinformatics and Genome Analysis/week 2 - NGS data QC and alignment in R/practical/MouseMamRNA-Seq/SRR1552452.fastq.gz.subread.BAM"
[10] "C:/Users/manso/OneDrive - University of West London/MSc Bioinformatics - UWL/6.BGA - Bioinformatics and Genome Analysis/week 2 - NGS data QC and alignment in R/practical/MouseMamRNA-Seq/SRR1552453.fastq.gz.subread.BAM"
[11] "C:/Users/manso/OneDrive - University of West London/MSc Bioinformatics - UWL/6.BGA - Bioinformatics and Genome Analysis/week 2 - NGS data QC and alignment in R/practical/MouseMamRNA-Seq/SRR1552454.fastq.gz.subread.BAM"
[12] "C:/Users/manso/OneDrive - University of West London/MSc Bioinformatics - UWL/6.BGA - Bioinformatics and Genome Analysis/week 2 - NGS data QC and alignment in R/practical/MouseMamRNA-Seq/SRR1552455.fastq.gz.subread.BAM"props <- propmapped(files=bam.files)
props
NumTotal NumMapped PropMapped
SRR1552444.fastq.gz.subread.BAM 1000 141 0.141
SRR1552445.fastq.gz.subread.BAM 1000 132 0.132
SRR1552446.fastq.gz.subread.BAM 1000 119 0.119
SRR1552447.fastq.gz.subread.BAM 1000 112 0.112
SRR1552448.fastq.gz.subread.BAM 1000 53 0.053
SRR1552449.fastq.gz.subread.BAM 1000 75 0.075
SRR1552450.fastq.gz.subread.BAM 1000 124 0.124
SRR1552451.fastq.gz.subread.BAM 1000 158 0.158
SRR1552452.fastq.gz.subread.BAM 1000 142 0.142
SRR1552453.fastq.gz.subread.BAM 1000 147 0.147
SRR1552454.fastq.gz.subread.BAM 1000 130 0.130
SRR1552455.fastq.gz.subread.BAM 1000 117 0.117As a result of the alignment, there are several BAM files, each containing read alignments for each library. BAM files contain the chromosomal location of each read.
9. Quality control after alignemnt
By using the qualityScores function in Rsubread, we can examine the quality scores associated with each base that has been called by the sequencing machine.
9.1. Extract quality scores(qs) for 100 reads
qs <- qualityScores(filename="C:/Users/manso/OneDrive - University of West London/MSc Bioinformatics - UWL/6.BGA - Bioinformatics and Genome Analysis/week 2 - NGS data QC and alignment in R/practical/MouseMamRNA-Seq/SRR1552450.fastq.gz",nreads=100)
qualityScores Rsubread 2.8.2
Scan the input file...
Totally 1000 reads were scanned; the sampling interval is 9.
Now extract read quality information...
Completed successfully. Quality scores for 100 reads (equally spaced in the file) are returned.9.2. Check dimension of qs
dim(qs)
[1] 100 1009.3. Check first few elements of qs with head:
head(qs)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
[1,] 31 34 31 37 37 37 37 37 39 37 39 39 39 41 40 41 41 41 36 38 40
[2,] 34 34 34 37 37 37 37 37 39 39 39 39 39 41 41 41 41 39 40 41 41
[3,] 34 34 31 37 37 37 37 37 39 39 39 39 39 41 41 41 41 41 41 41 41
[4,] 34 34 34 37 37 37 37 37 39 39 39 39 39 41 41 41 41 39 40 40 41
[5,] 34 34 34 37 37 37 37 37 39 39 39 39 39 41 41 41 40 41 41 41 41
[6,] 34 34 34 37 37 37 37 37 39 39 39 39 39 41 41 41 41 41 41 41 41
22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42
[1,] 41 39 40 38 40 40 40 40 40 38 40 40 40 38 37 39 40 40 41 40 41
[2,] 41 41 41 41 41 41 41 41 41 41 41 38 40 41 41 41 41 41 41 41 41
[3,] 41 41 41 41 41 41 41 41 40 40 41 38 38 38 40 41 40 41 39 40 40
[4,] 41 41 41 40 41 41 41 41 41 40 41 41 41 41 40 41 41 41 41 41 41
[5,] 41 41 40 41 41 41 41 41 39 40 41 41 41 41 40 41 41 41 41 41 41
[6,] 41 41 41 41 36 40 40 41 41 41 41 40 38 40 38 40 41 41 40 41 41
43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63
[1,] 41 38 40 40 40 41 41 41 40 40 36 40 38 37 37 35 35 33 35 35 33
[2,] 41 41 41 41 41 41 41 41 41 41 41 41 41 41 41 40 41 41 41 39 39
[3,] 41 41 41 39 40 38 40 40 37 39 40 41 41 40 37 39 39 39 37 37 37
[4,] 41 41 41 41 41 41 41 41 41 41 41 41 41 40 40 41 40 41 41 41 41
[5,] 41 41 41 41 41 41 41 41 41 40 41 41 41 41 40 41 41 41 38 40 41
[6,] 41 40 40 41 40 40 40 41 40 39 33 36 35 34 35 35 35 35 35 35 35
64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84
[1,] 35 34 35 35 35 34 34 34 35 35 35 35 35 35 35 36 35 35 35 34 34
[2,] 39 39 37 37 37 37 36 36 36 34 36 35 35 35 35 35 35 35 35 35 35
[3,] 37 37 37 36 36 35 36 35 33 35 35 35 35 34 36 35 35 34 35 35 35
[4,] 40 41 41 39 39 39 39 37 37 37 37 37 37 36 36 36 36 36 36 35 35
[5,] 41 41 41 39 39 39 39 39 38 37 37 37 37 37 35 35 35 35 35 36 35
[6,] 33 33 33 34 31 31 34 29 34 33 34 35 32 32 34 34 33 31 34 34 18
85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100
[1,] 35 34 35 35 35 35 35 35 35 35 35 35 35 35 35 33
[2,] 37 35 36 35 35 35 35 35 35 36 36 35 35 34 34 35
[3,] 34 35 35 35 35 35 33 35 35 35 31 34 35 35 33 33
[4,] 35 35 35 35 35 35 35 34 35 35 35 35 35 35 35 35
[5,] 35 35 35 35 35 35 35 35 35 36 35 35 35 35 35 35
[6,] 24 32 33 35 35 27 32 31 32 34 34 35 34 34 33 349.4. Visualise Quality scores
With a quality score of 30, the likelihood of making an incorrect call is 1 in 1000. (A quality score of 10 is a 1 in 10 chance of an incorrect base call.) A boxplot can be used to examine the overall distribution of quality scores across the 100 reads.
boxplot(qs)
10. Counting
The featureCounts() function can be used to calculate the number of mapped reads across mouse genes. There are built-in annotations for mouse genome assemblies (mm9, mm10) and human genome assemblies (hg19) (NCBI refseq annotations).
featureCounts() takes all the BAM files as input, and outputs an object which includes the count matrix. Each sample is a separate column, each row is a gene.
fc <- featureCounts(bam.files, annot.inbuilt="mm10")
NCBI RefSeq annotation for mm10 (build 38.1) is used.
========== _____ _ _ ____ _____ ______ _____
===== / ____| | | | _ \| __ \| ____| /\ | __ \
===== | (___ | | | | |_) | |__) | |__ / \ | | | |
==== \___ \| | | | _ <| _ /| __| / /\ \ | | | |
==== ____) | |__| | |_) | | \ \| |____ / ____ \| |__| |
========== |_____/ \____/|____/|_| \_\______/_/ \_\_____/
Rsubread 2.8.2
//========================== featureCounts setting ===========================\\
|| ||
|| Input files : 12 BAM files ||
|| ||
|| SRR1552444.fastq.gz.subread.BAM ||
|| SRR1552445.fastq.gz.subread.BAM ||
|| SRR1552446.fastq.gz.subread.BAM ||
|| SRR1552447.fastq.gz.subread.BAM ||
|| SRR1552448.fastq.gz.subread.BAM ||
|| SRR1552449.fastq.gz.subread.BAM ||
|| SRR1552450.fastq.gz.subread.BAM ||
|| SRR1552451.fastq.gz.subread.BAM ||
|| SRR1552452.fastq.gz.subread.BAM ||
|| SRR1552453.fastq.gz.subread.BAM ||
|| SRR1552454.fastq.gz.subread.BAM ||
|| SRR1552455.fastq.gz.subread.BAM ||
|| ||
|| Paired-end : no ||
|| Count read pairs : no ||
|| Annotation : inbuilt (mm10) ||
|| Dir for temp files : . ||
|| Threads : 1 ||
|| Level : meta-feature level ||
|| Multimapping reads : counted ||
|| Multi-overlapping reads : not counted ||
|| Min overlapping bases : 1 ||
|| ||
\\============================================================================//
//================================= Running ==================================\\
|| ||
|| Load annotation file mm10_RefSeq_exon.txt ... ||
|| Features : 222996 ||
|| Meta-features : 27179 ||
|| Chromosomes/contigs : 43 ||
|| ||
|| Process BAM file SRR1552444.fastq.gz.subread.BAM... ||
|| Single-end reads are included. ||
|| Total alignments : 1000 ||
|| Successfully assigned alignments : 2 (0.2%) ||
|| Running time : 0.00 minutes ||
|| ||
|| Process BAM file SRR1552445.fastq.gz.subread.BAM... ||
|| Single-end reads are included. ||
|| Total alignments : 1000 ||
|| Successfully assigned alignments : 5 (0.5%) ||
|| Running time : 0.00 minutes ||
|| ||
|| Process BAM file SRR1552446.fastq.gz.subread.BAM... ||
|| Single-end reads are included. ||
|| Total alignments : 1000 ||
|| Successfully assigned alignments : 4 (0.4%) ||
|| Running time : 0.00 minutes ||
|| ||
|| Process BAM file SRR1552447.fastq.gz.subread.BAM... ||
|| Single-end reads are included. ||
|| Total alignments : 1000 ||
|| Successfully assigned alignments : 1 (0.1%) ||
|| Running time : 0.00 minutes ||
|| ||
|| Process BAM file SRR1552448.fastq.gz.subread.BAM... ||
|| Single-end reads are included. ||
|| Total alignments : 1000 ||
|| Successfully assigned alignments : 0 (0.0%) ||
|| Running time : 0.00 minutes ||
|| ||
|| Process BAM file SRR1552449.fastq.gz.subread.BAM... ||
|| Single-end reads are included. ||
|| Total alignments : 1000 ||
|| Successfully assigned alignments : 3 (0.3%) ||
|| Running time : 0.00 minutes ||
|| ||
|| Process BAM file SRR1552450.fastq.gz.subread.BAM... ||
|| Single-end reads are included. ||
|| Total alignments : 1000 ||
|| Successfully assigned alignments : 6 (0.6%) ||
|| Running time : 0.00 minutes ||
|| ||
|| Process BAM file SRR1552451.fastq.gz.subread.BAM... ||
|| Single-end reads are included. ||
|| Total alignments : 1000 ||
|| Successfully assigned alignments : 2 (0.2%) ||
|| Running time : 0.00 minutes ||
|| ||
|| Process BAM file SRR1552452.fastq.gz.subread.BAM... ||
|| Single-end reads are included. ||
|| Total alignments : 1000 ||
|| Successfully assigned alignments : 2 (0.2%) ||
|| Running time : 0.00 minutes ||
|| ||
|| Process BAM file SRR1552453.fastq.gz.subread.BAM... ||
|| Single-end reads are included. ||
|| Total alignments : 1000 ||
|| Successfully assigned alignments : 8 (0.8%) ||
|| Running time : 0.00 minutes ||
|| ||
|| Process BAM file SRR1552454.fastq.gz.subread.BAM... ||
|| Single-end reads are included. ||
|| Total alignments : 1000 ||
|| Successfully assigned alignments : 1 (0.1%) ||
|| Running time : 0.00 minutes ||
|| ||
|| Process BAM file SRR1552455.fastq.gz.subread.BAM... ||
|| Single-end reads are included. ||
|| Total alignments : 1000 ||
|| Successfully assigned alignments : 5 (0.5%) ||
|| Running time : 0.00 minutes ||
|| ||
|| Write the final count table. ||
|| Write the read assignment summary. ||
|| ||
\\============================================================================//names(fc) #See what slots are stored in fc
[1] "counts" "annotation" "targets" "stat" “fc$stat” shows the statistics of the read mapping, also reporting the unassigned reads and the reasons why they weren’t assigned:
fc$stat
Status SRR1552444.fastq.gz.subread.BAM
1 Assigned 2
2 Unassigned_Unmapped 859
3 Unassigned_Read_Type 0
4 Unassigned_Singleton 0
5 Unassigned_MappingQuality 0
6 Unassigned_Chimera 0
7 Unassigned_FragmentLength 0
8 Unassigned_Duplicate 0
9 Unassigned_MultiMapping 0
10 Unassigned_Secondary 0
11 Unassigned_NonSplit 0
12 Unassigned_NoFeatures 138
13 Unassigned_Overlapping_Length 0
14 Unassigned_Ambiguity 1
SRR1552445.fastq.gz.subread.BAM SRR1552446.fastq.gz.subread.BAM
1 5 4
2 868 881
3 0 0
4 0 0
5 0 0
6 0 0
7 0 0
8 0 0
9 0 0
10 0 0
11 0 0
12 127 115
13 0 0
14 0 0
SRR1552447.fastq.gz.subread.BAM SRR1552448.fastq.gz.subread.BAM
1 1 0
2 888 947
3 0 0
4 0 0
5 0 0
6 0 0
7 0 0
8 0 0
9 0 0
10 0 0
11 0 0
12 111 53
13 0 0
14 0 0
SRR1552449.fastq.gz.subread.BAM SRR1552450.fastq.gz.subread.BAM
1 3 6
2 925 876
3 0 0
4 0 0
5 0 0
6 0 0
7 0 0
8 0 0
9 0 0
10 0 0
11 0 0
12 71 117
13 0 0
14 1 1
SRR1552451.fastq.gz.subread.BAM SRR1552452.fastq.gz.subread.BAM
1 2 2
2 842 858
3 0 0
4 0 0
5 0 0
6 0 0
7 0 0
8 0 0
9 0 0
10 0 0
11 0 0
12 156 140
13 0 0
14 0 0
SRR1552453.fastq.gz.subread.BAM SRR1552454.fastq.gz.subread.BAM
1 8 1
2 853 870
3 0 0
4 0 0
5 0 0
6 0 0
7 0 0
8 0 0
9 0 0
10 0 0
11 0 0
12 139 129
13 0 0
14 0 0
SRR1552455.fastq.gz.subread.BAM
1 5
2 883
3 0
4 0
5 0
6 0
7 0
8 0
9 0
10 0
11 0
12 112
13 0
14 0In this matrix, the rows denote the Entrez gene identifiers for each gene, and the columns denote the output filenames from the align function.
FeatureCounts() compiled reads over genes using annotation information in the annotation slot.
head(fc$annotation)
GeneID Chr
1 497097 chr1;chr1;chr1
2 100503874 chr1;chr1
3 100038431 chr1
4 19888 chr1;chr1;chr1;chr1;chr1;chr1
5 20671 chr1;chr1;chr1;chr1;chr1
6 27395 chr1;chr1;chr1;chr1;chr1
Start
1 3214482;3421702;3670552
2 3647309;3658847
3 3680155
4 4290846;4343507;4351910;4352202;4360200;4409170
5 4490928;4493100;4493772;4495136;4496291
6 4773198;4777525;4782568;4783951;4785573
End Strand Length
1 3216968;3421901;3671498 -;-;- 3634
2 3650509;3658904 -;- 3259
3 3681788 + 1634
4 4293012;4350091;4352081;4352837;4360314;4409241 -;-;-;-;-;- 9747
5 4492668;4493466;4493863;4495942;4496413 -;-;-;-;- 3130
6 4776801;4777648;4782733;4784105;4785726 -;-;-;-;- 4203