Hey guys, I am helping a coworker to build a fungal phylogenetic tree.
In brief, the sequences were amplified by EF4/FUNG5 from ocean environmental DNA. Clone libraries were constructed and the sequences were screened and trimmed. OTUs were grouped at 3% cutoff. References were determined by AFTOL-BLAST. My sequences and references were imported into MEGA5 for Muscle alignment. The alignment was manually corrected and trimmed. RaxmlGUI1.3.1 was used to build the tree. Most of my sequences were assigned to Pezizomycotina. Zygomycota was used as outgroup.
I uploaded the tree file. As you can see, it looks really odd. The uncommon deep branch is hard to resolve. I also uploaded the alignment. Hope someone can help.
I've done some similar work before but this time it's totally weird.
Kind. Fang
Maria is right, some of the sequences are in the wrong orientation.
In your alignment (in MEGA) you should look for a highly conserved region, that is identical in most of the sequences. If you then use the search function to mark a fragment of 10-15 bases, you will see it highlighted in almost all the sequences. Those that do not show this mark are probably in the wrong orientation.
I tried position 333-346 for a search and rev.complemented every sequence that did not show the mark-up and it worked.
Try using the ARB or similar software, it is usually good to have a reference tree to know where you are going.
http://www.arb-home.de/
V-RevComp supports batch-processing of large 16S/18S datasets in terms of reverse complementary detection:
https://www.researchgate.net/publication/229523581_VREVCOMP_automated_highthroughput_detection_of_reverse_complementary_16S_rRNA_gene_sequences_in_large_environmental_and_taxonomic_datasets?ev=srch_pub
Article V-REVCOMP: Automated high-throughput detection of reverse co...
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