hello everyone,
I have been using Bowtie2 for Illumina 100x2 RNA-Seq datasets. I understand TopHat was built as Bowtie (older version) couldn't do gapped alignment. Now that Bowtie2 does that, what is the status of TopHat usage?
Kindly advice. I have followed this strategy ->
Bowtie2 --> SAM/ BAM ---> Cufflinks (with GTF file) ---> transcripts with FPKM
Till now for the 8 datasets processed, I obtained ~99% alignment with "proper-paired" @ ~85%.
What am I missing by not using TopHat? Any suggestions or ideas, please..
---Bowtie2 STDOUT for one of the datasets ---
Time loading reference: 00:00:08
Time loading forward index: 00:00:19
Time loading mirror index: 00:00:11
Multiseed full-index search: 15:44:57
70363764 reads; of these:
70363764 (100.00%) were paired; of these:
8612578 (12.24%) aligned concordantly 0 times
34458617 (48.97%) aligned concordantly exactly 1 time
27292569 (38.79%) aligned concordantly >1 times
----
8612578 pairs aligned concordantly 0 times; of these:
5036749 (58.48%) aligned discordantly 1 time
----
3575829 pairs aligned 0 times concordantly or discordantly; of these:
7151658 mates make up the pairs; of these:
1238386 (17.32%) aligned 0 times
2331211 (32.60%) aligned exactly 1 time
3582061 (50.09%) aligned >1 times
99.12% overall alignment rate
Time searching: 15:45:35
Overall time: 15:45:35
I have been using Bowtie2 for Illumina 100x2 RNA-Seq datasets. I understand TopHat was built as Bowtie (older version) couldn't do gapped alignment. Now that Bowtie2 does that, what is the status of TopHat usage?
- Would it be right to align using Bowtie2 and reach Cufflinks directly?
- What would I be missing if I don't use TopHat but the new Bowtie?
Kindly advice. I have followed this strategy ->
Bowtie2 --> SAM/ BAM ---> Cufflinks (with GTF file) ---> transcripts with FPKM
Till now for the 8 datasets processed, I obtained ~99% alignment with "proper-paired" @ ~85%.
What am I missing by not using TopHat? Any suggestions or ideas, please..
---Bowtie2 STDOUT for one of the datasets ---
Time loading reference: 00:00:08
Time loading forward index: 00:00:19
Time loading mirror index: 00:00:11
Multiseed full-index search: 15:44:57
70363764 reads; of these:
70363764 (100.00%) were paired; of these:
8612578 (12.24%) aligned concordantly 0 times
34458617 (48.97%) aligned concordantly exactly 1 time
27292569 (38.79%) aligned concordantly >1 times
----
8612578 pairs aligned concordantly 0 times; of these:
5036749 (58.48%) aligned discordantly 1 time
----
3575829 pairs aligned 0 times concordantly or discordantly; of these:
7151658 mates make up the pairs; of these:
1238386 (17.32%) aligned 0 times
2331211 (32.60%) aligned exactly 1 time
3582061 (50.09%) aligned >1 times
99.12% overall alignment rate
Time searching: 15:45:35
Overall time: 15:45:35
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