# Compare Samples With Sequences of Different Lengths? **URL:** \ **Category:** Commands in mothur\ **Created:** [August 25, 2025, 8:04am UTC](https://forum.mothur.org/t/compare-samples-with-sequences-of-different-lengths/22360 "2025-08-25T08:04:10Z")\ **Posts on this page:** 5\ **Page:** 1
**Author:** ![brick1233](https://avatars.discourse-cdn.com/v4/letter/b/edb3f5/32.png) [@brick1233](https://forum.mothur.org/u/brick1233)\ **Post date:** [August 25, 2025, 8:04am UTC](https://forum.mothur.org/t/compare-samples-with-sequences-of-different-lengths/22360/1 "2025-08-25T08:04:10Z")
Hello, this post is mainly a question about how to compare samples from different experiments/16S regions. I’m trying to conduct a meta-analysis of different studies looking at the same biological process but with different designs. The experiments use 3 different regions: V3-V4 (n=2), V4-V5 (n=1), and V4 (n=1). My approach has been so far to follow the MiSeq SOP for each dataset, and then try to merge the sequences/taxonomy abundances post-pipeline. I’m using PICRUSt2 to do functional predictions, and plan on binning the functional abundances based on the respective OTUs’ genus level classifications, an approach that I think is imperfect but “good enough”. However, I would also like to compare the community structure of samples from different experiments. Does anyone have an idea on how I could do this? Feel free to critique my methods as well, this is my first time making a pipeline like this. vr, 🫡 ---
**Author:** ![pschloss](https://yyz2.discourse-cdn.com/flex036/user_avatar/forum.mothur.org/pschloss/32/4_2.png) [@pschloss](https://forum.mothur.org/u/pschloss)\ **Post date:** [August 25, 2025, 12:46pm UTC](https://forum.mothur.org/t/compare-samples-with-sequences-of-different-lengths/22360/2 "2025-08-25T12:46:36Z")
Hi there - I would analyze those regions separately, conduct your analysis separately, and then pool the results. Generally I discourage this type of analysis since it splits your N unnecessarily. Keep in mind that each primer set and region has it’s own biases, strengths, and weaknesses. If the n=# is the number of samples you have, I don’t know what type of analysis you’ll really be able to perform since those numbers - even if pooled - are quite small. Here is an example of how we have done this in the past… [https://journals.asm.org/doi/10.1128/mbio.01018-16](https://journals.asm.org/doi/10.1128/mbio.01018-16) As another critique… I wouldn’t bother with picrust. The genes and functions you’ll get back are largely generic. Also, metagenomic analysis for this type of work is somewhat suspect. If you have one taxon increase/decrease in abundance, then all of its 4000 genes will also increase/decrease. Which one is the most important? Pretty impossible to say IMHO. Pat ---
**Author:** ![brick1233](https://avatars.discourse-cdn.com/v4/letter/b/edb3f5/32.png) [@brick1233](https://forum.mothur.org/u/brick1233)\ **Post date:** [August 25, 2025, 1:29pm UTC](https://forum.mothur.org/t/compare-samples-with-sequences-of-different-lengths/22360/3 "2025-08-25T13:29:24Z")
Thanks for the feedback, @pschloss. That’s pretty much what I expected, and I agree with your remark about PICRUSt. I will definitely emphasize the limitations of this approach in the report I write. From what I have found with the available data certainly makes your [blog post](https://mothur.org/blog/2014/Why-such-a-large-distance-matrix) still relevant. I do not want that “grimy feeling that makes you want to take a bath”. May I use your response as a reference? All the best, thanks again. edit: I’m only really focused on ~20 genes from the CAZy family, so it’s not like I’m looking for the needle in a hay stack. The key for me is how the communities’ functions (eg. cellulase/pectinase “abundances”) are correlated to specific points in the process. Does this make the implementation of PICRUSt2 slightly more relevant? ---
**Author:** ![pschloss](https://yyz2.discourse-cdn.com/flex036/user_avatar/forum.mothur.org/pschloss/32/4_2.png) [@pschloss](https://forum.mothur.org/u/pschloss)\ **Post date:** [August 25, 2025, 1:48pm UTC](https://forum.mothur.org/t/compare-samples-with-sequences-of-different-lengths/22360/4 "2025-08-25T13:48:04Z")
No problem - feel free to use me as a reference 🙂 ---
**Author:** ![system](https://yyz2.discourse-cdn.com/flex036/user_avatar/forum.mothur.org/system/32/2_2.png) [@system](https://forum.mothur.org/u/system)\ **Post date:** [September 4, 2025, 1:48pm UTC](https://forum.mothur.org/t/compare-samples-with-sequences-of-different-lengths/22360/5 "2025-09-04T13:48:54Z")
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