# Mapping with STAR ## Execute ``` cd ~/LSLNGS2015/ mkdir RNASEQ_data/star_GM12878_rep1 RNASEQ_data/star_GM12878_rep2 STAR --genomeDir GENOME_data/star --readFilesCommand zcat \ --readFilesIn RNASEQ_data/GM12878.rep1.R1.fastq.gz RNASEQ_data/GM12878.rep1.R2.fastq.gz \ --outSAMtype BAM SortedByCoordinate --limitBAMsortRAM 16000000000 --outSAMunmapped Within \ --twopassMode Basic --outFilterMultimapNmax 1 --quantMode TranscriptomeSAM \ --runThreadN 20 --outFileNamePrefix "RNASEQ_data/star_GM12878_rep1/" STAR --genomeDir GENOME_data/star --readFilesCommand zcat \ --readFilesIn RNASEQ_data/GM12878.rep2.R1.fastq.gz RNASEQ_data/GM12878.rep2.R2.fastq.gz \ --outSAMtype BAM SortedByCoordinate --limitBAMsortRAM 16000000000 --outSAMunmapped Within \ --twopassMode Basic --outFilterMultimapNmax 1 --quantMode TranscriptomeSAM \ --runThreadN 20 --outFileNamePrefix "RNASEQ_data/star_GM12878_rep2/" mkdir RNASEQ_data/star_K562_rep1 RNASEQ_data/star_K562_rep2 STAR --genomeDir GENOME_data/star --readFilesCommand zcat \ --readFilesIn RNASEQ_data/K562.rep1.R1.fastq.gz RNASEQ_data/K562.rep1.R2.fastq.gz \ --outSAMtype BAM SortedByCoordinate --limitBAMsortRAM 16000000000 --outSAMunmapped Within \ --twopassMode Basic --outFilterMultimapNmax 1 --quantMode TranscriptomeSAM \ --runThreadN 20 --outFileNamePrefix "RNASEQ_data/star_K562_rep1/" STAR --genomeDir GENOME_data/star --readFilesCommand zcat \ --readFilesIn RNASEQ_data/K562.rep2.R1.fastq.gz RNASEQ_data/K562.rep2.R2.fastq.gz \ --outSAMtype BAM SortedByCoordinate --limitBAMsortRAM 16000000000 --outSAMunmapped Within \ --twopassMode Basic --outFilterMultimapNmax 1 --quantMode TranscriptomeSAM \ --runThreadN 20 --outFileNamePrefix "RNASEQ_data/star_K562_rep2/" ``` ## Resource usage | Sample | ALPS Queue Name | CPU Time | Max Memory | Duration | | ------- | --------------- | -------------- | ---------- | ---------------------------------- | | GM12878 | 128G | 123358.00 sec. | 40 GB | 3 hours 6 minutes 59 seconds | | K562 | 128G | 110262.00 sec. | 40 Gb | 3 hours, 49 minutes and 52 seconds | ## Options `--genomeDir` path to the directory where genome files are stored. `--sjdbGTFfile` skip this if provided during database creation step. `--readFilesIn` paths to files that contain input read1 (and read2 if PE sequencing). `--readFilesCommand` command line to execute for each of the input file. For example: `zcat` to uncompress .gz files. `--outSAMtype` type of output, i.e. SAM or BAM. `--outFilterMultimapNmax` read alignments will be output only if the read maps fewer than this value, otherwise no alignments will be output. Default is 10. `--outSAMunmapped` output of unmapped reads in the SAM format, None or Within SAM file. `--quantMode` types of quantification requested, i.e. GeneCounts and/or TranscriptomeSAM. `--twopassMode` 2-pass mapping mode. In the first pass, the novel junctions are detected and inserted into the genome indices. In the second pass, all reads will be re-mapped using annotated (from the GTF file) and novel (detected in the first pass) junctions. While this doubles the run time, it significantly increases sensitivity to novel splice junctions. `--runThreadN` number of threads to run STAR. `--outFileNamePrefix` output files name prefix. ## Take a look at the STAR alignment files generated `ls -la ~/LSLNGS2015/RNASEQ_data/star_GM12878_rep1/` ``` -rw-rw-r-- 1 ycl6 ycl6 15995801460 Oct 25 12:57 Aligned.sortedByCoord.out.bam -rw-rw-r-- 1 ycl6 ycl6 20352373974 Oct 25 12:36 Aligned.toTranscriptome.out.bam -rw-rw-r-- 1 ycl6 ycl6 1866 Oct 25 12:57 Log.final.out -rw-rw-r-- 1 ycl6 ycl6 27784 Oct 25 12:57 Log.out -rw-rw-r-- 1 ycl6 ycl6 6111 Oct 25 12:57 Log.progress.out -rw-rw-r-- 1 ycl6 ycl6 8360503 Oct 25 12:57 SJ.out.tab drwx------ 2 ycl6 ycl6 4096 Oct 25 11:42 _STARgenome drwx------ 2 ycl6 ycl6 4096 Oct 25 12:00 _STARpass1 ``` ## Simple statistics with samtools flagstat `samtools flagstat star_GM12878_rep1/Aligned.sortedByCoord.out.bam` ``` 210178300 + 0 in total (QC-passed reads + QC-failed reads) 0 + 0 secondary 0 + 0 supplementary 0 + 0 duplicates 190658902 + 0 mapped (90.71% : N/A) 210178300 + 0 paired in sequencing 105089150 + 0 read1 105089150 + 0 read2 190658902 + 0 properly paired (90.71% : N/A) 190658902 + 0 with itself and mate mapped 0 + 0 singletons (0.00% : N/A) 0 + 0 with mate mapped to a different chr 0 + 0 with mate mapped to a different chr (mapQ>=5) ``` ## Alignment report The `Log.final.out` shows the mapping statistics, it is very useful for quality control. The statistics are calculated for each read (single- or paired-end) and then summed or averaged over all reads. STAR counts a paired-end read as one read. Most of the information is collected about the UNIQUE mappers. Each splicing is counted in the numbers of splices, and will correspond to summing the counts in `SJ.out.tab`. The mismatch/indel error rates are calculated on a per base basis, i.e. as total number of mismatches/indels in all unique mappers divided by the total number of mapped bases. `cat ~/LSLNGS2015/RNASEQ_data/star_GM12878_rep1/Log.final.out` ``` Started job on | Oct 25 11:38:22 Started mapping on | Oct 25 12:03:39 Finished on | Oct 25 12:57:53 Mapping speed, Million of reads per hour | 116.26 Number of input reads | 105089150 Average input read length | 202 UNIQUE READS: Uniquely mapped reads number | 95329451 Uniquely mapped reads % | 90.71% Average mapped length | 200.93 Number of splices: Total | 42610775 Number of splices: Annotated (sjdb) | 42438693 Number of splices: GT/AG | 42035836 Number of splices: GC/AG | 373883 Number of splices: AT/AC | 42947 Number of splices: Non-canonical | 158109 Mismatch rate per base, % | 0.29% Deletion rate per base | 0.02% Deletion average length | 1.57 Insertion rate per base | 0.01% Insertion average length | 1.49 MULTI-MAPPING READS: Number of reads mapped to multiple loci | 0 % of reads mapped to multiple loci | 0.00% Number of reads mapped to too many loci | 7822366 % of reads mapped to too many loci | 7.44% UNMAPPED READS: % of reads unmapped: too many mismatches | 0.00% % of reads unmapped: too short | 1.82% % of reads unmapped: other | 0.02% CHIMERIC READS: Number of chimeric reads | 0 % of chimeric reads | 0.00% ``` ## Splice junctions `SJ.out.tab` contains high confidence collapsed splice junctions in tab-delimited format. STAR defines the junction start/end as intronic bases, other software may define them as exonic bases. The columns have the following meaning: | Column | Description | | ------ | ------------------------------------------------------------------------------------------- | | 1 | chromosome | | 2 | first base of the intron (1-based) | | 3 | last base of the intron (1-based) | | 4 | strand (0: undefined, 1: +, 2: -) | | 5 | intron motif (0: non-canonical, 1: GT/AG, 2: CT/AC, 3: GC/AG, 4: CT/GC, 5: AT/AC, 6: GT/AT) | | 6 | 0: unannotated, 1: annotated (only if splice junctions database is used) | | 7 | number of uniquely mapping reads crossing the junction | | 8 | number of multi-mapping reads crossing the junction | | 9 | maximum spliced alignment overhang | `head ~/LSLNGS2015/RNASEQ_data/star_GM12878_rep1/SJ.out.tab` ``` 1 15039 15795 2 2 1 10 0 35 1 15247 185761 0 0 1 12 0 37 1 16607 186518 2 4 1 4 0 26 1 16766 16857 2 2 1 27 0 42 1 17056 17232 2 2 1 15 0 25 1 17056 17525 2 2 1 2 0 42 1 17056 17914 2 2 1 4 0 19 1 17369 17605 2 2 1 62 0 50 1 17530 187839 0 0 1 4 0 31 1 17743 17914 2 2 1 61 0 50 ``` --- # Agent Instructions: Querying This Documentation If you need additional information that is not directly available in this page, you can query the documentation dynamically by asking a question. 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