RNA Seq Read Representation by Trinity Assembly

Brian Haas edited this page Feb 1, 2018 · 17 revisions

Assessing the Read Content of the Transcriptome Assembly

Assembled transcripts might not always fully represent properly paired-end reads, as some transcripts may be fragmented or short and only one fragment read of a pair may align. Simply aligning reads to your transcriptome assembly using bowtie or STAR will only capture the properly paired reads. To assess the read composition of our assembly, we want to capture and count all reads that map to our assembled transcripts, including the properly paired and those that are not.

In order to comprehensively capture read alignments, we run the process below. Bowtie2 is used to align the reads to the transcriptome and then we count the number of proper pairs and improper or orphan read alignments.

First, build a bowtie2 index for the transcriptome:

bowtie2-build Trinity.fasta Trinity.fasta

Then perform the alignment (example for paired-end reads) to just capture the read alignment statistics.

bowtie2 -p 10 -q --no-unal -k 20 -x Trinity.fasta -1 reads_1.fq -2 reads_2.fq  \
     | samtools view -@10 -Sb -o bowtie2.bam 2>&1 | tee align_stats.txt

.

76201190 reads; of these:
  76201190 (100.00%) were paired; of these:
    18166307 (23.84%) aligned concordantly 0 times
    17026716 (22.34%) aligned concordantly exactly 1 time
    41008167 (53.82%) aligned concordantly >1 times
    ----
    18166307 pairs aligned concordantly 0 times; of these:
      1769907 (9.74%) aligned discordantly 1 time
    ----
    16396400 pairs aligned 0 times concordantly or discordantly; of these:
      32792800 mates make up the pairs; of these:
        15287552 (46.62%) aligned 0 times
        3874965 (11.82%) aligned exactly 1 time
        13630283 (41.56%) aligned >1 times
89.97% overall alignment rate

Note, if you want to actually capture the aligned reads into a file, you can adjust your command accordingly, but in the above usage, we're simply interested in the mapping statistics alone. Also, with the above parameters, even though multiple read mappings are counted in the report above, they may not be output into a resulting bam file. Examine your bowtie2 parameter usage to determine what will be reported in the alignment output file if you do wish to retain these alignments.

A typical Trinity transcriptome assembly will have the vast majority of all reads mapping back to the assembly, and ~70-80% of the mapped fragments found mapped as proper pairs (yielding concordant alignments 1 or more times to the reconstructed transcriptome).

Visualize read support using IGV

The Integrative Genomics Viewer is useful for visualizing read support across any of the Trinity assemblies. The bowtie2 alignments generated above, which are currently sorted by read name, can be re-sorted according to coordinate, indexed, and then viewed along with the Trinity assemblies using the IGV browser as follows.

# sort the alignments by coordinate
samtools sort bowtie2.bam -o bowtie2.coordSorted.bam

# index the coordinate-sorted bam file
samtools index bowtie2.coordSorted.bam

# index the Trinity.fasta file
samtools faidx Trinity.fasta

# view the aligned reads along the Trinity assembly reference contigs.
# note, you can do this by using the various graphical menu options in IGV (load genome 'Trinity.fasta', load file 'bowtie2.coordSorted.bam'), or you can use the command-line tool like so:

igv.sh -g `pwd`/Trinity.fasta  `pwd`/bowtie2.coordSorted.bam

Note, the above assumes that the Trinity.fasta and bowtie2.coordSorted.bam files are in your current working directory.

And you can then go to any Trinity assembly of interest and examine the read (and paired-end) support. An example region of a long Trinity transcript contig is shown below.

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