# Need a second opinion on my data - DADA2, truncation length for V4 region - 250 bases PE read data
**URL:** https://forum.qiime2.org/t/need-a-second-opinion-on-my-data-dada2-truncation-length-for-v4-region-250-bases-pe-read-data/4401
**Category:** User Support
**Tags:** dada2, truncate
**Created:** [June 2, 2018, 8:02am UTC](https://forum.qiime2.org/t/need-a-second-opinion-on-my-data-dada2-truncation-length-for-v4-region-250-bases-pe-read-data/4401 "2018-06-02T08:02:40Z")
**Posts on this page:** 5
**Page:** 1
### Author:  [@anwesh](https://forum.qiime2.org/u/anwesh)
#### Post date: [June 2, 2018, 8:02am UTC](https://forum.qiime2.org/t/need-a-second-opinion-on-my-data-dada2-truncation-length-for-v4-region-250-bases-pe-read-data/4401/1 "2018-06-02T08:02:40Z")
Hi all,
I know this question has been asked earlier, but I just want to confirm that I'm doing it right for my dataset, so, please input your suggestions...
Here are the details and workflow...
**Dataset Properties:** 7 stool samples, 16S rDNA - V4 region, 2\*250bp, HiSeq platform, Demultiplexed
Quality checking with **FASTQC** :
**_Sample1\_R1_**
**_Sample1\_R2_**
* * *
Used **Trimmomatic** to remove adaptor content and filter out low quality reads...
```
$ java -jar trimmomatic-0.38.jar PE -phred33 FJ1_R1.fastq.gz FJ1_R2.fastq.gz FJ1_for_paired.fq.gz FJ1_for_unpaired.fq.gz FJ1_rev_paired.fq.gz FJ1_rev_unpaired.fq.gz ILLUMINACLIP:TruSeq3-PE-2.fa:2:30:10 LEADING:20 TRAILING:20 SLIDINGWINDOW:4:20 MINLEN:100
```
* * *
The paired forward and reverse reads were imported into **qiime2 (qiime2-2018.4)**
```
$ qiime tools import --type 'SampleData[PairedEndSequencesWithQuality]' --input-path jar_manifest.csv --output-path raw_imports/jar-raw.qza --source-format PairedEndFastqManifestPhred33
```
* * *
Quality visualization of the imported raw reads with qiime tools...
* * *
Further, **DADA2** was used for denoising with two different truncation values...
1. trunc-len-f 200 and trunc-len-r 175
```
$ qiime dada2 denoise-paired --i-demultiplexed-seqs jar-raw.qza --p-trunc-len-f 200 --p-trunc-len-r 175 --p-chimera-method pooled --p-n-threads 8 --o-representative-sequences denoise/jar_rep-seqs.qza --o-table denoise/jar_table.qza --o-denoising-stats denoise/jar_stats.qza --verbose
```
Output is [jar\_table.qzv](https://cdck-file-uploads-global.s3.dualstack.us-west-2.amazonaws.com/flex002/uploads/qiime21/original/2X/5/5f31eb18308c09f3cf3f7f440bedbac23e0b7216.qzv) (366.8 KB)
Total features: 1342
Total frequency: 831,346
2. No truncation, as the quality was comparatively better:
```
$ qiime dada2 denoise-paired --i-demultiplexed-seqs jar-raw.qza --p-trunc-len-f 0 --p-trunc-len-r 0 --p-chimera-method pooled --p-n-threads 8 --o-representative-sequences denoise/jar-untr_rep-seqs.qza --o-table denoise/jar-untr_table.qza --o-denoising-stats denoise/jar-untr_stats.qza --verbose
```
Output is [jar-untr\_table.qzv](https://cdck-file-uploads-global.s3.dualstack.us-west-2.amazonaws.com/flex002/uploads/qiime21/original/2X/e/ece97f977ed7086b6625207c891c902d0c9cf552.qzv) (339.3 KB)
Total features: 699
Total frequencies: 2,428,146
* * *
I understood from this [topic](https://forum.qiime2.org/t/dada2-truncation-lengths-and-features-number/1940/6) that the changes in total features and frequencies are based on the probability of finding the error-free reads...
Now my questions are...
1. Is the workflow correct? Using trimmomatic prior to denoising?
2. Does the **`MINLEN`** parameter of Trimmomatic effect the total output of the reads? Do I need to increase it to ~175 (end of good quality in reverse reads)?
3. **DADA2:** As it is clear that truncation is required, are the values used for truncation appropriate?
4. Do I still need to truncate the reverse reads? _(I can run it and check, but, with the computational resources availble here, it is taking a minimum of 72 hrs to complete the denoising for this dataset)_
5. Whether I need to trim the 5` end of the reverse reads?
Thank you for your time...
With Regards
Anwesh
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### Author:  [@thermokarst](https://forum.qiime2.org/u/thermokarst)
#### Post date: [June 4, 2018, 10:37pm UTC](https://forum.qiime2.org/t/need-a-second-opinion-on-my-data-dada2-truncation-length-for-v4-region-250-bases-pe-read-data/4401/2 "2018-06-04T22:37:22Z")
---
### Author:  [@thermokarst](https://forum.qiime2.org/u/thermokarst)
#### Post date: [June 4, 2018, 10:42pm UTC](https://forum.qiime2.org/t/need-a-second-opinion-on-my-data-dada2-truncation-length-for-v4-region-250-bases-pe-read-data/4401/3 "2018-06-04T22:42:17Z")
Hi @anwesh!
> [@anwesh](#):
>
> Is the workflow correct? Using trimmomatic prior to denoising?
I know you mentioned computational resources are limited, but maybe it is worth processing a subset of your samples with and without this trimmomatic step so that you can compare the results.
> [@anwesh](#):
>
> Does the **`MINLEN`** parameter of Trimmomatic effect the total output of the reads? Do I need to increase it to ~175 (end of good quality in reverse reads)?
I don't know, we don't develop trimmomatic, but I suspect that this parameter does impact the final number of reads.
> [@anwesh](#):
>
> **DADA2:** As it is clear that truncation is required, are the values used for truncation appropriate?
They look reasonable to me, but again, maybe it is worth testing out a few sets of values on a subset of samples and comparing the results.
> [@anwesh](#):
>
> Do I still need to truncate the reverse reads?
It looks like you truncated your reverse reads in example 1 above.
> [@anwesh](#):
>
> Whether I need to trim the 5` end of the reverse reads?
Maybe! Again, I would recommend testing this out on a subset of your samples to get an understanding for how these parameters will impact your downstream results.
Hope that helps! 🦖 :qiime2:
---
### Author:  [@thermokarst](https://forum.qiime2.org/u/thermokarst)
#### Post date: [June 4, 2018, 10:42pm UTC](https://forum.qiime2.org/t/need-a-second-opinion-on-my-data-dada2-truncation-length-for-v4-region-250-bases-pe-read-data/4401/4 "2018-06-04T22:42:19Z")
---
### Author:  [@system](https://forum.qiime2.org/u/system)
#### Post date: [July 7, 2018, 11:41am UTC](https://forum.qiime2.org/t/need-a-second-opinion-on-my-data-dada2-truncation-length-for-v4-region-250-bases-pe-read-data/4401/6 "2018-07-07T11:41:31Z")
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