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 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

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