How/when to merge replicates

Nis_12 · October 17, 2019, 5:43am

Dear sir,

I am doing metagenome analysis of microbiome. please give me the details that we have to remove barcode or not.

Mehrbod_Estaki · October 19, 2019, 4:33pm

Hi @Nis_12,
We generally don’t answer questions via DM, instead please post your question in a new public thread that way answers can be used by others in the future.
I would also advise to provide much more details about your specific question and what you are trying to do. Thanks!

Edit: This post was originally in response to a private message that has since been reclassified as a “public topic”.

Nis_12 · October 21, 2019, 5:16am

sir i am handling 16S rna data for metagenome analysis. as i am bit confused that i have i biological event with 3 replicates. each replicates have their own barcode sequence. As we are trying to merge the replicates. after merging the replicates which barcodes we have chhoosed?

colinbrislawn · October 22, 2019, 5:15pm

Hello @Nis_12,

Welcome to the Qiime 2 forums!

I would recommend keeping the replicates separate.

How many samples to you have in total?

Colin

Nis_12 · October 23, 2019, 7:06am

I have two years dataset and its around 75 samples

Nis_12 · October 23, 2019, 7:08am

Dear sir,
as my guide advice to merge the replicates and i am bit confused how to identify the the dataset that is it emp or casava type sequence.
please help me out these problems

ben · October 23, 2019, 3:16pm

Likely EMP, but you should try both

Nis_12 · October 24, 2019, 5:39am

Dear @ben
But my guide suggest me to run Casava. then how we decide is it emp or casava ?

ben · October 24, 2019, 1:58pm

Take a quick screenshot of the mapping file w/ barcodes. Also, could you in terminal run:

head yourfastq.file.fastq

Then post the results here. Ben

Nis_12 · October 25, 2019, 7:13am

Dear @ben
I am not able to underatand. Which file you were saying?

Nis_12 · October 25, 2019, 7:18am

Dear @colinbrislawn If i merged the sequence then wat barcode we should select.

ben · October 25, 2019, 1:26pm

The file that contains the 75 samples. What is it? Ben

Nis_12 · October 28, 2019, 5:52am

dear @ben its is sff file. i had convert sff to fastq

ben · October 28, 2019, 1:38pm

Great, I am unfamiliar w/ the format, but it appears to be a 454 pyrosequencing format. You can try to import it that way, can you screenshot the series of files? Ben

colinbrislawn · October 28, 2019, 5:30pm

You could import using all your barcodes, than after that you could choose to merge samples.

I would encourage you to keep all your samples separate.

Nis_12 · October 29, 2019, 5:28am

yes. these are the format of files

ben · October 29, 2019, 2:49pm

I would encourage as @colinbrislawn has said to not merge the samples. There is no straightforward way without manipulation of the files (e.g., editing from a *txt file -> adding sequence counts). This is unsupported and discouraged that you do this.

My question for you: Why do you want to merge these files? Are there some features that are found in each file individually? Do you need to examine the merge file for this reason? There is a way to do this. but again, highly discouraged as this is manipulating the actual data. Ben

Nis_12 · October 30, 2019, 7:51am

Dear @ben
As i have number of samples and i have to compare the three sets of data. so we try to make it simpler and easier through merge the files.

ben · October 30, 2019, 11:41am

I'm going to apologize, but I am going to assume to put these samples into one QIIME artifact (.qza) file?

Can I see how the files look as fastq? You want to import multiple files that were Pyrosequencing -> Fastq -> QIIME2 artifact?

Once you made the fastq files it looks like you should do what this user did:

gzip the entire fastq folder into a fastq.gz
import it using a manifest method

Ben

Nis_12 · October 31, 2019, 4:43am

i also have import the qza file