Examine Strand Specificity

Brian Haas edited this page Feb 7, 2017 · 4 revisions
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Examine Strand Specificity of RNA-Seq Reads

You an estimate the level of strand-specificity of your RNA-Seq data by aligning the reads back to your Trinity assembly and examining the distribution of RNA-Seq read (or fragment) orientations on those assemblies. You can also readily detect whether you've assembled data as non-strand-specific but the reads are actually strand-specific. You can this like so:

First, align your reads back against your Trinity assembly. Bowtie2 works well for this:

  ${TRINITY_HOME}/util/misc/run_bowtie2.pl  Trinity.fasta  left.fq right.fq \
         | samtools view -Sb - | samtools sort - -o bowtie2.coordSorted.bam

Then, examine the distribution of strand-specificity - looking at the distribution of orientations for the first read of paired-end fragment reads.

   ${TRINITY_HOME}/util/misc/examine_strand_specificity.pl bowtie2.coordSorted.bam

This will generate a data table and a pdf plot showing the distribution of the ratio computed for each contig:

     (plus_strand - minus_strand) / total

based on the orientation of the first read of each RNA-Seq fragment.

Example data tables and corresponding plots are shown below for different data configurations, including strand-specific data, non-strand-specific data, and then the case where strand-specific data were assembled as non-strand-specific (oops!).

Non-strand-specific RNA-Seq data

Example data table:

#transcript               plus_strand_1stReads  minus_strand_1stReads  total_reads  diff_ratio
TRINITY_DN4743_c2_g9_i2   67251                 75742                  142993       -0.059
TRINITY_DN3140_c0_g2_i1   30326                 32768                  63094        -0.039
TRINITY_DN4633_c1_g3_i1   28114                 26549                  54663        0.029
TRINITY_DN4382_c0_g1_i1   19946                 20993                  40939        -0.026
TRINITY_DN4566_c0_g2_i1   20232                 18057                  38289        0.057
TRINITY_DN1432_c0_g1_i1   16724                 18159                  34883        -0.041
TRINITY_DN4888_c0_g1_i1   15508                 16726                  32234        -0.038
TRINITY_DN4744_c5_g3_i1   14500                 16904                  31404        -0.077
TRINITY_DN4733_c6_g21_i1  15967                 14721                  30688        0.041
TRINITY_DN4736_c4_g9_i1   15188                 15257                  30445        -0.002
TRINITY_DN5011_c0_g1_i1   12907                 13673                  26580        -0.029

and the 'diff_ratio' values plotted according to top cumulative quantiles of total numbers of reads.

Strand specificity (dUTP approach, --SS_lib_type RF)

Example data table:

#transcript                 plus_strand_1stReads  minus_strand_1stReads  total_reads  diff_ratio
TRINITY_DN14418_c1_g5_i1    31                    98783                  98814        -0.999
TRINITY_DN13820_c0_g1_i1    30                    80623                  80653        -0.999
TRINITY_DN14453_c6_g4_i2    9                     75834                  75843        -1.000
TRINITY_DN14080_c15_g1_i1   6                     68692                  68698        -1.000
TRINITY_DN14331_c10_g3_i2   14                    68234                  68248        -1.000
TRINITY_DN14453_c6_g4_i1    14                    62573                  62587        -1.000
TRINITY_DN14138_c0_g1_i2    9                     56515                  56524        -1.000
TRINITY_DN14120_c1_g1_i1    3                     51368                  51371        -1.000
TRINITY_DN14020_c0_g1_i2    4                     43347                  43351        -1.000
TRINITY_DN14081_c1_g1_i1    27                    41332                  41359        -0.999
TRINITY_DN13058_c0_g1_i1    592                   38763                  39355        -0.970

Strand-specific RNA-Seq, but assembled as non-strand-specific

#transcript                 plus_strand_1stReads  minus_strand_1stReads  total_reads  diff_ratio
TRINITY_DN13653_c2_g1_i1    93806                 22                     93828        1.000
TRINITY_DN13640_c9_g3_i3    69431                 14                     69445        1.000
TRINITY_DN13631_c3_g1_i1    66033                 11                     66044        1.000
TRINITY_DN13652_c2_g2_i2    60368                 7                      60375        1.000
TRINITY_DN13315_c9_g1_i1    6                     50921                  50927        -1.000
TRINITY_DN13637_c7_g1_i1    46489                 4                      46493        1.000
TRINITY_DN13631_c3_g1_i2    42728                 6                      42734        1.000
TRINITY_DN13560_c7_g6_i1    8                     41954                  41962        -1.000
TRINITY_DN13057_c0_g3_i1    827                   40913                  41740        -0.960
TRINITY_DN13563_c8_g3_i1    41339                 29                     41368        0.999
TRINITY_DN12884_c0_g1_i2    21                    40477                  40498        -0.999
TRINITY_DN12884_c0_g1_i1    11                    40365                  40376        -0.999
TRINITY_DN13630_c2_g3_i1    39921                 4                      39925        1.000
TRINITY_DN13093_c1_g1_i2    3                     39072                  39075        -1.000
TRINITY_DN13653_c2_g3_i1    38485                 39                     38524        0.998