I hope this message finds you well. I am currently conducting a meta-analysis involving various studies and am in the process of compiling all the data for 16S rRNA analysis using Mothur. I’m reaching out to seek your expertise on a couple of questions I have encountered during the process.
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Data Trimming with PCR.seqs:
I am working with two types of read data: single reads and paired reads. The paired reads cover the V3-V4 regions, while the single reads cover V3 and V4 separately. After aligning this data with the V3-V4 SILVA database, I’ve observed the following:- Paired reads align from positions 1 to 18897 (440 bp).
- A portion of single reads aligns from positions 1 to 7481, and most align from positions 6737 to 18897.
Given this alignment pattern, can I use the pcr.seqs command to trim the aligned files specifically from position 6737 to 18897? I aim to standardize both the paired and single reads for further analysis.
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Filtering Challenges:
I encountered an issue where applying the filter.seqs command with the trump= option resulted in the deletion of all data. However, when proceeding without this step, I am unable to obtain a phylogenetic bootstrap. I am considering using alternative tools like MEGA12 to overcome this. Do you have recommendations on managing this filtering step or insights into the implications of using a different tool to achieve accurate phylogenetic analysis?
Your guidance on these matters would be greatly appreciated. I am eager to ensure the integrity and reliability of the analysis and look forward to any advice you might have.