Question: Trimmomatic output results in extremely low alignment using bowtie2
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gravatar for danielryan9287
9 months ago by
danielryan92870 wrote:

Hello, I have trimmed by paired end data RNA seq (Illumina Miseq) data using trimmomatic. However after aligning with bowtie2, I get virtually noalignments. Please see below:

6953839 reads; of these: 6953839 (100.00%) were paired; of these: 6951481 (99.97%) aligned concordantly 0 times 2357 (0.03%) aligned concordantly exactly 1 time 1 (0.00%) aligned concordantly >1 times ---- 6951481 pairs aligned concordantly 0 times; of these: 38758 (0.56%) aligned discordantly 1 time ---- 6912723 pairs aligned 0 times concordantly or discordantly; of these: 13825446 mates make up the pairs; of these: 13825088 (100.00%) aligned 0 times 196 (0.00%) aligned exactly 1 time 162 (0.00%) aligned >1 times 0.59% overall alignment rate

However if I perform the alignment of the original fastq files (not trimmed), I get high alignments. See below:

7656360 reads; of these: 7656360 (100.00%) were paired; of these: 1097101 (14.33%) aligned concordantly 0 times 6195514 (80.92%) aligned concordantly exactly 1 time 363745 (4.75%) aligned concordantly >1 times ---- 1097101 pairs aligned concordantly 0 times; of these: 295377 (26.92%) aligned discordantly 1 time ---- 801724 pairs aligned 0 times concordantly or discordantly; of these: 1603448 mates make up the pairs; of these: 1203571 (75.06%) aligned 0 times 351300 (21.91%) aligned exactly 1 time 48577 (3.03%) aligned >1 times 92.14% overall alignment rate

Please let me know a possible reason for this?

Thanks.

alignment bowtie2 trimmomatic • 490 views
ADD COMMENTlink modified 9 months ago by Jennifer Hillman Jackson25k • written 9 months ago by danielryan92870
0
gravatar for Jennifer Hillman Jackson
9 months ago by
United States
Jennifer Hillman Jackson25k wrote:

Hello,

The QA was probably too aggressive. Run FastQC on the fasta data before and after QA steps to tune the trimming parameters to better fit your data.

Tutorials: https://galaxyproject.org/learn/

Start with this one: NGS logistics - this is an introduction to Galaxy's functionality for the analysis of Next Generation Sequencing data.

Thanks! Jen, Galaxy team

ADD COMMENTlink modified 9 months ago • written 9 months ago by Jennifer Hillman Jackson25k
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