No archaea despite universal primers

[Sabrin (Abdelghany)]

I have the situation that, I used universal primers 341F and 806R for V3-V4 region. However, in my taxa Barplots, I have only bacteria but no archaea at all. Any suggestion what would be the reason for that?

@SoilRotifer, Here is the steps I used to train my classifier following [RESCRIPt] , Have I done anything in my followed steps that I only targeted bacteria?

Option 2: Install within QIIME 2 environment

• conda install -c conda-forge -c bioconda -c qiime2 -c defaults xmltodict
• pip install git+https://github.com/bokulich-lab/RESCRIPt.git

qiime rescript get-silva-data --p-version '138' --p-target 'SSURef_NR99' --p-include-species-labels --o-silva-sequences silva-138-ssu-nr99-rna-seqs.qza --o-silva-taxonomy silva-138-ssu-nr99-tax.qza

qiime rescript reverse-transcribe --i-rna-sequences silva-138-ssu-nr99-rna-seqs.qza --o-dna-sequences silva-138-ssu-nr99-seqs.qza

“Culling” low-quality sequences with cull-seqs

time qiime rescript cull-seqs --i-sequences silva-138-ssu-nr99-seqs.qza --p-n-jobs 5 --o-clean-sequences silva-138-ssu-nr99-seqs-cleaned.qza
Filtering sequences by length and taxonomy

time qiime rescript filter-seqs-length-by-taxon --i-sequences silva-138-ssu-nr99-seqs-cleaned.qza --i-taxonomy silva-138-ssu-nr99-tax.qza --p-labels Archaea Bacteria Eukaryota --p-min-lens 900 1200 1400 --o-filtered-seqs silva-138-ssu-nr99-seqs-filt.qza --o-discarded-seqs silva-138-ssu-nr99-seqs-discard.qza
Dereplicating in uniq mode

time qiime rescript dereplicate --i-sequences silva-138-ssu-nr99-seqs-filt.qza --i-taxa silva-138-ssu-nr99-tax.qza --p-rank-handles 'silva' --p-mode 'uniq' --o-dereplicated-sequences silva-138-ssu-nr99-seqs-derep-uniq.qza --o-dereplicated-taxa silva-138-ssu-nr99-tax-derep-uniq.qza

Make amplicon-region specific classifier
qiime feature-classifier extract-reads \

*--i-sequences silva-138-ssu-nr99-seqs-derep-uniq.qza *

*--p-f-primer CCTACGGGAGGCAGCAG *

*--p-r-primer GGACTACHVGGGTWTCTAAT *

*--p-n-jobs 2 *

*--p-read-orientation 'forward' *

--o-reads silva-138-ssu-nr99-seqs-341f -806r .qza

Dereplicate extracted region

time qiime rescript dereplicate --i-sequences silva-138-ssu-nr99-seqs-341 f-806r.qza --i-taxa silva-138-ssu-nr99-tax-derep-uniq.qza --p-rank-handles 'silva' --p-mode 'uniq' --o-dereplicated-sequences silva-138-ssu-nr99-seqs- 341 f-806r-uniq.qza --o-dereplicated-taxa silva-138-ssu-nr99-tax- 341f -806r-derep-uniq.qza

build our amplicon-region specific classifier.
qiime feature -classifier fit -classifier-naive -bayes
--i-reference-reads silva-138-ssu-nr99-seqs-341f-806r-uniq.qza --i-reference-taxonomy silva-138-ssu-nr99-tax-341f-806r-derep-uniq.qza --o-classifier silva-138-ssu-nr99-341f-806r-classifier.qza

Looking forward for your kind guidance

Here is my taxa barplots attached
atra-taxa-bar-plots.qzv (833.5 KB)

[SoilRotifer (Mike Robeson)]

Hi @Sabrin,

It looks like your classifier was constructed properly. I've made a similar V3V4 classifier myself and there are indeed Archaea reference sequences within the V3V4 classifier (16,470 of them to be exact). Are you expecting there to be Archaea within your samples?

You can check which taxa are in your V3V4 taxonomy file (prior to making the classifier) by making a visualizer:

qiime metadata tabulate \
    --m-input-file silva-138-ssu-nr99-tax-341f-806r-derep-uniq.qza \
    --o-visualization silva-138-ssu-nr99-tax-341f-806r-derep-uniq.qzv

Then in the visualization, up at the top right, type Archaea.

-Mike

[Sabrin (Abdelghany)]

Thank you for your support.

Yes classifier looks good, it has archaea included but strange to me why my universal primers amplified no archaea at all. It is gut microbiome in marine environment as well as water and sediment samples

[SoilRotifer (Mike Robeson)]

@Sabrin

strange to me why my universal primers amplified no archaea at all.

It could simply be that there are no archaea in your samples, or, they are in such low abundance that they are not being detected / amplified. Likely due to the abundances of other taxa.

There can be many reasons for this, often sequencing depth can be a factor in detecting low-abundant taxa, even primer biases.

[Mechah]

Hi Sabrin,
do you have any prior knowledge about the expected abundances of archaea (e.g. from qPCR) in your samples? - or at least qualitative estimates from targeted cultivation, FISH etc.? Which archaeal taxa do you expect to find in your samples - methanogens, ammonia oxidizing archaea, halophiles etc.? While it is hard to cover all archaeal phyla with one primer setup it might be easier to target specific archaeal taxa (if you know what to look for). Our lab is specialized for human-associated archaea and it is often tricky and also biased if you use common universal primers for the detection alone. Hence, it is easy to overlook them. Here is a publication related to this topic: Frontiers | Exploring the Archaeome: Detection of Archaeal Signatures in the Human Body
I would recommend to use an archaea specific approach for qPCR and an amplicon analysis (be aware that you should not make any quantitative assessements at all if you use a nested amplicon approach alone...).