the weird microbial composition when using different library preparation kit

Hello, have a good time. I am really new in qiime2.
I have trouble with combining a dataset which were made by 5 sequencing runs. Two sequcing runs use the Truseq nano library preparation kit and the others using nextera library preparation kit.

I run my microbiome pipeline starting from demultiplexed fastq, however when I check the abundance table on phylum level, D_0_Bacteria;_ has higher abundance in the samples using Truseq nano library preparation kit. In contrast, the samples with nextera library preparation kit have much lower abundance of D_0_Bacteria;_ .

my questions are:
(1) how to combine the datasets using different library preparation
(2) how to eliminate bias between each sequencing run
(3) why the D_0_Bacteria;_ has extremely different between two library preparation kit
Could you give me some advices on how to face those situations?

Thanks a lot


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Hi @Chih-Ying,

Welcome to the :qiime2: forum!

I can't speak to the differences in Nexera and Treseq nano specifically, but kit effects are a pretty well known issue. You might want to look at the MBQC paper which discusses specific issues related to kit effects. You might also be interested in past contaminant removal threads, although I think this is maybe less relevant:

Harmonization is a lot harder, and depends on how you plan to analyze your data. You might look at the perc-norm plugin, which may help with some of the normalization, although I'm not sure how you integrate this across analysis.

In other cases, it really depends on what analysis you're planning to do.

Does the nextera kit have more Archea? Or just more unclassified reads?
Were there any other differences in parameters beteen the two kits?


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