unlike intergenic sequences genes are highly conserved throughout many lifeforms so contamination may not be the only answer. Your results may depend on the quality of your sequencing and possibly in your amplified gene just a couple of poorly called bases or even taq error basecalls could mean that blast finds homology to other species if they are quite similar. Attention to the annealing temperature of the pcr ( as high as possible ) and negative controls ( in case of contamination) and manual trimming of the poorly sequenced ends of the pcr product ( if using Sanger sequencing) may make a difference to what BLAST finds. Did you get a good strong signal and very low background signal on the electropherogram of your sequencing?

Hello Paul Rutland

Thank you for your answer and feedback, it seems to very helpful for me

About your question : Yes, I get a good signal for the amplification process as validation for the sequencing process (file attached in this comment)

FYI

Sequencing result (BLAST) from 8 sampel shown as

1. C. marulioides -> 93%

2. C. marulioides -> 100%

3. Osphronemus goramy -> 93%

4. Osphronemus goramy -> 93%

5. C. narulioides -> 98%

6. Pythium sp. -> 78%

7. Pythium sp. -> 78%

8. Pythium sp. -> 78%

Notes:

Universal COI primer

a. sample 1-5 looks clear

b. sample 6-8 not visible

Primer HCO2198-LCO1490

a. sample 6-8 clearly visible

So I first used COI universal primer for samples 1-5, and the rest used HCO2198-LCO1490

Hello Faiz Irvan .The amplification looks a bit weak and I am still concerned about the strength of the signal generated by the sequencing reaction. Can you attach an original .abi sequence file from the sequencing company for one of the sequences that is being miscalled as bacterium please ( also one good sequence file for comparison please)

Of course sir

NB:

1st_BASE_4032829_2_PCO1 --> Good sequences (Channa)

1st_BASE_4032834_8_PLCO --> Bacterium

1st_BASE_4032830_4_PCO1 --> goramy (Other species)