# How to do multiplex of Ion torrent sequences
**URL:** https://forum.qiime2.org/t/how-to-do-multiplex-of-ion-torrent-sequences/2907
**Category:** User Support
**Tags:** import
**Created:** [February 6, 2018, 5:06pm UTC](https://forum.qiime2.org/t/how-to-do-multiplex-of-ion-torrent-sequences/2907 "2018-02-06T17:06:20Z")
**Posts on this page:** 20
**Page:** 1
### Author:  [@khemlalnirmalkar](https://forum.qiime2.org/u/khemlalnirmalkar)
#### Post date: [February 6, 2018, 5:06pm UTC](https://forum.qiime2.org/t/how-to-do-multiplex-of-ion-torrent-sequences/2907/1 "2018-02-06T17:06:20Z")
Dear Qiime2 users,
I am newbie to bioinformatic analysis and for qiime2 as well. We are using Ion torrent PGM platform for V4 16SrRNA region. After sequencing, output are all .fastq files (according to our samples) and one nomatch.fastq, kindly see the attachment (
. It seems our sequencer itself do demultiplex. My doubts are -
1. As I understood in qiime2 tutorial, we need barcodes.fastq file but i could not able to find the same file in our output.
2. I could not able to understand this barcodes.fastq file. Inside the file how the barcodes and sample sequences are arranged.
3. My nomatch\_fastq file (as in attachment) looks like barcodes.fastq file but i am not sure is the same file to process further or do I need to look for something else.
Kindly help me to understand qiime2 tutorial.
Thanks,
Regards,
Khemlal
---
### Author:  [@colinbrislawn](https://forum.qiime2.org/u/colinbrislawn)
#### Post date: [February 6, 2018, 5:13pm UTC](https://forum.qiime2.org/t/how-to-do-multiplex-of-ion-torrent-sequences/2907/2 "2018-02-06T17:13:49Z")
Hello Khemala,
Thanks for posting and getting started with Qiime!
Do you have one fastq file per sample? If so, your data should be a good fit for the [fastq manifest format](https://docs.qiime2.org/2017.12/tutorials/importing/#fastq-manifest-formats). That page discribes the format, and has a few examples of import commands.
Let me know if that command looks like a good for your data.
Keep us posted,
Colin
---
### Author:  [@khemlalnirmalkar](https://forum.qiime2.org/u/khemlalnirmalkar)
#### Post date: [February 12, 2018, 3:40pm UTC](https://forum.qiime2.org/t/how-to-do-multiplex-of-ion-torrent-sequences/2907/3 "2018-02-12T15:40:57Z")
Hello Colin,
Thanks for your reply.
I checked the " **fastq manifest format**" and as I understood this command is not gonaa work with my datas. Yes, I have one fastq file per sample please see the attachment **fastq.png**"
" but I don't have forward and reverse sequences separately. Each fastq data has data like please see the attachment **F8\_fastq.png**" ". I was looking the Qiime2 tutorial and it seems like this tutorial is for illumina sequences and in my case I am using Ion Torrent PGM and output is not same like illumina. I dont have barcode.fastq file and I am not able to proceed further.
Kindly help me to solve this issue.
Thanks,
Sincerely,
Khemlal
---
### Author:  [@colinbrislawn](https://forum.qiime2.org/u/colinbrislawn)
#### Post date: [February 12, 2018, 4:56pm UTC](https://forum.qiime2.org/t/how-to-do-multiplex-of-ion-torrent-sequences/2907/5 "2018-02-12T16:56:23Z")
Good morning Khemala,
Thanks for posting your files for me. I think this format will work for importing: [Single End Fastq Manifest Phred33](https://docs.qiime2.org/2017.12/tutorials/importing/#singleendfastqmanifestphred33). This format only needs a forward sequences, which is a perfect fit for your data. So when the tutorial says `reverse`, you just don't include those lines.
> [@khemlalnirmalkar](#):
>
> I was looking the Qiime2 tutorial and it seems like this tutorial is for illumina sequences and in my case I am using Ion Torrent PGM and output is not same like illumina. I don't have barcode.fastq file and I am not able to proceed further.
Yes, a lot of the qiime tutorials are about Illumina. We should write some tutorials for Ion Torrent data too. The good news is that after you import your data and error correct it, the rest of analysis is exactly the same.
Colin
---
### Author:  [@khemlalnirmalkar](https://forum.qiime2.org/u/khemlalnirmalkar)
#### Post date: [February 13, 2018, 12:18am UTC](https://forum.qiime2.org/t/how-to-do-multiplex-of-ion-torrent-sequences/2907/6 "2018-02-13T00:18:28Z")
Hello Colin,
Good Morning,
Thanks for your useful comments. I am sorry, I didn't explain properly in my previous post. Here in attachment "
" explains the outline of Ion Torrent PGM platform. So we get the merged FASTQ files from forward and reverse reads. In previous post's image " "
the name like **V3-341F8.fastq** etc. where **"F8"** or all other files like from **"F8- F46".fastq** are just barcode numbers to identify the files.
I hope now I made clear myself regarding my problem with Qiime2 to use the tutorial for Ion Torrent PGM platform based sequences. Kindly give your suggestion how to start the process.
Thanks,
Sincerely,
Khemlal
---
### Author:  [@colinbrislawn](https://forum.qiime2.org/u/colinbrislawn)
#### Post date: [February 13, 2018, 4:19pm UTC](https://forum.qiime2.org/t/how-to-do-multiplex-of-ion-torrent-sequences/2907/7 "2018-02-13T16:19:32Z")
Good morning Khemlal,
That's a very helpful diagram! I'm not very familiar with the Ion Torrent PFM platform, but here is what I would try first.
1. Build your manifest file and save it as `se-33-manifest.csv`. Maybe like this:
```auto
sample-id,absolute-filepath,direction
# Lines starting with '#' are ignored and can be used to create
# "comments" or even "comment out" entries
sample-8,$PWD/some/filepath/V3-341F8-.fastq,forward
sample-9,$PWD/some/filepath/V3-341F9-.fastq,forward
sample-10,$PWD/some/filepath/V3-341F10-.fastq,forward
...
more samples
...
sample-46,$PWD/some/filepath/V3-341F46-.fastq,forward
```
1. Then import your reads:
```bash
qiime tools import \
--type 'SampleData[SequencesWithQuality]' \
--input-path se-33-manifest.csv \
--output-path single-end-demux.qza \
--source-format SingleEndFastqManifestPhred33
```
Let me know how well that process works. One key question for me is how the Ion Torrent goes about 'merging' the forward and reverse reads. This could change our demultiplexing strategy.
Colin
---
### Author:  [@thermokarst](https://forum.qiime2.org/u/thermokarst)
#### Post date: [February 13, 2018, 4:22pm UTC](https://forum.qiime2.org/t/how-to-do-multiplex-of-ion-torrent-sequences/2907/9 "2018-02-13T16:22:16Z")
Thanks for that @colinbrislawn!
We probably want to import as type `SampleData[JoinedSequencesWithQuality]`, instead:
```bash
qiime tools import \
--type 'SampleData[JoinedSequencesWithQuality]' \
--input-path se-33-manifest.csv \
--output-path single-end-demux.qza \
--source-format SingleEndFastqManifestPhred33
```
This will let QIIME 2 know that the reads are joined, instead of just single-end reads.
---
### Author:  [@MMC\_northS](https://forum.qiime2.org/u/MMC_northS)
#### Post date: [March 5, 2018, 3:02pm UTC](https://forum.qiime2.org/t/how-to-do-multiplex-of-ion-torrent-sequences/2907/10 "2018-03-05T15:02:00Z")
I am working with demultiplexed data from PGM Ion Torrent, like khemlalnirmalkar user, and I think that I understood how to import the data through the manifest file.csv (until my understanding a file with a list of all your fastq files paths.
In your example all reads are in forward in the column direction, however, the problem in PGM is that you can add the barcodes after the PCR, in which, the PCR product is 'blundt ends' so the barcodes can fixed in forward or reverse and you can have the reads in both directions in the same file, forward and reverse.
Is this a problem when you work with qiime?
---
### Author:  [@Nicholas\_Bokulich](https://forum.qiime2.org/u/Nicholas_Bokulich)
#### Post date: [March 5, 2018, 3:02pm UTC](https://forum.qiime2.org/t/how-to-do-multiplex-of-ion-torrent-sequences/2907/11 "2018-03-05T15:02:37Z")
---
### Author:  [@thermokarst](https://forum.qiime2.org/u/thermokarst)
#### Post date: [March 6, 2018, 12:28am UTC](https://forum.qiime2.org/t/how-to-do-multiplex-of-ion-torrent-sequences/2907/12 "2018-03-06T00:28:06Z")
The "direction" doesn't necessarily matter in that manifest, so long as it is all one, or all the other (forward or reverse) when importing single-end reads. Since you have demultiplexed reads, go ahead and import them as single-end, then [trim the barcodes using `q2-cutadapt`](https://docs.qiime2.org/2018.2/plugins/available/cutadapt/trim-paired/). You might need to set up a few parameters in that step in order to catch the barcodes in both directions. Let us know how that goes for you! 🦖
---
### Author:  [@thermokarst](https://forum.qiime2.org/u/thermokarst)
#### Post date: [March 7, 2018, 3:01pm UTC](https://forum.qiime2.org/t/how-to-do-multiplex-of-ion-torrent-sequences/2907/14 "2018-03-07T15:01:04Z")
An off-topic reply has been split into a new topic: [Help formatting FASTQ manifest file](https://forum.qiime2.org/t/help-formatting-fastq-manifest-file/3337)
Please keep replies on-topic in the future.
---
### Author:  [@khemlalnirmalkar](https://forum.qiime2.org/u/khemlalnirmalkar)
#### Post date: [April 5, 2018, 2:53pm UTC](https://forum.qiime2.org/t/how-to-do-multiplex-of-ion-torrent-sequences/2907/15 "2018-04-05T14:53:07Z")
Thanks for your comments @thermokarst @colinbrislawn @MMC_northS . In the second figure from my previous post, files from V3-341F8 to V3-341F46.fastq are containing both forward and reverse sequences.
I am sorry but I am bit confused. Since i don't have barcodes.fastq. file and I am worrying how to merge my all different sample .fastq file to create Biom file.
As we were doing in qiime1, the first fastq to fasta (fna) file then ,otus picking n biom file.
Thanks
---
### Author:  [@ebolyen](https://forum.qiime2.org/u/ebolyen)
#### Post date: [April 5, 2018, 3:00pm UTC](https://forum.qiime2.org/t/how-to-do-multiplex-of-ion-torrent-sequences/2907/16 "2018-04-05T15:00:30Z")
---
### Author:  [@thermokarst](https://forum.qiime2.org/u/thermokarst)
#### Post date: [April 10, 2018, 3:08pm UTC](https://forum.qiime2.org/t/how-to-do-multiplex-of-ion-torrent-sequences/2907/17 "2018-04-10T15:08:40Z")
Hi @khemlalnirmalkar -
If your reads are already demultiplexed, there is no need to worry about barcodes (just make sure they have been removed from your demultiplexed reads). Please see [my post above](https://forum.qiime2.org/t/how-to-do-multiplex-of-ion-torrent-sequences/2907/12) about importing these demultiplexed reads.
Since these reads are already technically "joined", you won't be able to use q2-dada2 (since that plugin requires your reads be unjoined), and you are also not able to use q2-deblur, since that tool only works with Illumina products. That leaves you with [OTU clustering](https://forum.qiime2.org/t/clustering-sequences-into-otus-using-q2-vsearch/1348) as the only available method for generating a feature table.
I hope that helps - let us know if you have any questions. Thanks!
---
### Author:  [@khemlalnirmalkar](https://forum.qiime2.org/u/khemlalnirmalkar)
#### Post date: [April 21, 2018, 1:25am UTC](https://forum.qiime2.org/t/how-to-do-multiplex-of-ion-torrent-sequences/2907/19 "2018-04-21T01:25:22Z")
Hello Qiime2 users @Nicholas_Bokulich @thermokarst @colinbrislawn @MMC_northS
As per your suggestions, again tried to import my .fna files (demultiflexed) using
**qiime tools import \ --input-path seqs.fna \ --output-path seqs.qza \ --type SampleData[Sequences]**
for the next step but I could not do successfully. It asked two kind of files. one .fna files and second barcode.fastq file. And again I got stuck in the first step as Ion Torrent doesn't provide barcode.fastq file.
@MMC_northS As you mentioned earlier, you are also working with Ion Torrent PGM, can you explain in detail, how did you process your .fastq files after sequencing?
Thanks,
Sincerely,
Khemlal
---
### Author:  [@MMC\_northS](https://forum.qiime2.org/u/MMC_northS)
#### Post date: [April 23, 2018, 8:09am UTC](https://forum.qiime2.org/t/how-to-do-multiplex-of-ion-torrent-sequences/2907/20 "2018-04-23T08:09:29Z")
Hi @khemlalnirmalkar
I have imported in QIIME2 my fastq files from PGM using the next command line:
qiime tools import
--type 'SampleData[SequencesWithQuality]'
--input-path manifest\_file.csv
--output-path demux.qza
--source-format SingleEndFastqManifestPhred33
You have the instructions to make your manifest\_file here: [Importing data — QIIME 2 2018.2.0 documentation](https://docs.qiime2.org/2018.2/tutorials/importing/) in the section "Fastq manifest formats".
After import your data you can continue doing analyses in QIIME2 (filter quality, clustering, denoising, assign taxonomy, etc....). Other option is use other program like Cutadapt to filter by quality and length, etc.) and then import the fastq filtered instead the raw files.
I hope this information will be useful for you 🙂
Good luck!
MMC
---
### Author:  [@thermokarst](https://forum.qiime2.org/u/thermokarst)
#### Post date: [April 23, 2018, 3:49pm UTC](https://forum.qiime2.org/t/how-to-do-multiplex-of-ion-torrent-sequences/2907/21 "2018-04-23T15:49:38Z")
---
### Author:  [@khemlalnirmalkar](https://forum.qiime2.org/u/khemlalnirmalkar)
#### Post date: [April 24, 2018, 1:27am UTC](https://forum.qiime2.org/t/how-to-do-multiplex-of-ion-torrent-sequences/2907/26 "2018-04-24T01:27:47Z")
Hello @MMC_northS Thanks for your suggestion. I tried as you mentioned and got an error as in the attached picture.
while importing my raw sequences (fastq files), I used --type option with EMPSingleEndSequences, but couldn't able to convert them. I tried some more file type but same error came.
I suppose my manifest.csv was correct "attached image manifest.jpg "
Kindly check and help me to correct my error.
Thanks,
Sincerely,
Khemlal
---
### Author:  [@MMC\_northS](https://forum.qiime2.org/u/MMC_northS)
#### Post date: [April 24, 2018, 6:32am UTC](https://forum.qiime2.org/t/how-to-do-multiplex-of-ion-torrent-sequences/2907/27 "2018-04-24T06:32:21Z")
Hi @khemlalnirmalkar
the first thing different to my command that I can see is in the --type command.
I used: --type ‘SampleData[SequencesWithQuality]’
and you used EMPSingleEnd Sequences ; and you did not use any symbol like '[........]'
Regarding your manifest file I supposed is right but it depending on where you have your sequences.fastq located in your computer or server.
I copy one line of my mapping file for just in case you can compare with yours.
Sample1,$PWD/Fastq\_for\_QIIME2/Sample1.fastq,forward
I used the absolute path or my working directory in that moment.
I hope you fix your error!
Best,
MMC
---
### Author:  [@antgonza](https://forum.qiime2.org/u/antgonza)
#### Post date: [April 24, 2018, 1:51pm UTC](https://forum.qiime2.org/t/how-to-do-multiplex-of-ion-torrent-sequences/2907/28 "2018-04-24T13:51:33Z")
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