importing fasta file from PacBio

binzuo · December 4, 2019, 6:00pm

Hello, I've got a dataset sequenced by PacBio Single Molecular Real-Time, which is a fasta file.
I want to apply qiime2 for the analysis, and based on my search here, dada2 1.10, which is integrated in qiime2-2019.10, should support this full-length results. Please correct me if I am wrong.

The problem I have is importing the data. I know there are so many formats that qiime2 supports, but I don't know which one is for this one. The head of the file looks like this.

U1A_1
GACGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAACGAACTCTGGTATTGATTGGTGCTTGCATCATGATTTACATTTGAGTGAGTGGCGAACTGGTGAGTAACACGTGGGAAACCTGCCCAGAAGCGGGGGATAACACCTGGAAACAGATGCTAATACCGCATAACAACTTGGACCGCATGGTCCGAGTTTGAAAGATGGCTTCGGCTATCACTTTTGGATGGTCCCGCGGCGTATTAGCTAGATGGTGAGGTAACGGCTCACCATGGCAATGATACGTAGCCGACCTGAGAGGGTAATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGAAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAACTCTGTTGTTAAAGAAGAACATATCTGAGAGTAACTGTTCAGGTATTGACGGTATTTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTTTTAAGTCTGATGTGAAAGCCTTCGGCTCAACCGAAGAAGTGCATCGGAAACTGGGAAACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGTATGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCATACCGTAAACGATGAATGCTAAGTGTTGGAGGGTTTCCGCCCTTCAGTGCTGCAGCTAACGCATTAAGCATTCCGCCTGGGGAGTACGGCCGCAAGGCTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGACATACTATGCAAATCTAAGAGATTAGACGTTCCCTTCGGGGACATGGATACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTATCAGTTGCCAGCATTAAGTTGGGCACTCTGGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGTGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAACGAGTTGCGAACTCGCGAGAGTAAGCTAATCTCTTAAAGCCATTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGTCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTGTAACACCCAAAGTCGGTGGGGTAACCTTTTAGGAACCAGCCGCCTAAGGTGGGACAGATGATTAGGGTG
L5B_2
ATTGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAACGGTAACAGGAAGCAGCTTGCTGTTTCGCTGACGAGTGGCGGACGGGTGAGTAATGTCTGGGAAACTGCCTGATGGAGGGGATAACTACTGGAAACGGTAGCTAATACCGCATAACGTCGCAAGACCAAAGAGGGGGACCTTCGGGCCTCTTGCCATCGGATGTGCCCAGATGGGATTAGCTAGTAGGTGGGGTAACGGCTCACCTAGGCGACGATCCCTAGCTGGTCTGAGAGGATGACCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCCATGCCGCGTGTATGAAGAAGGCCTTCGGGTTGTAAAGTACTTTCAGCGGGAGGAAGGGAGTAAAGTTAATACCTTTGCTCATTGACGTTACCCGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCACGCAGGCGGTTTGTTAAGTCAGATGTGAAATCCCGGGCTCAACCTGGGAACTGCATCTGATACTGGCAAGCTTGAGTCTCGTAGAGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACGAAGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCGACTTGGAGGTTGTGCCCTTGAGGCGTGGCTTCCGGAGCTAACGCGTTAAGTCGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTGGTCTTGACATCCACGGAAGTTTCAGAGATGGAATGTGCCTTCGGGAACCGTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATCCTTTGTTGCCAGCGGTCCGGCCGGGAACTCAAAGGAGACTGCCAGTGATAAACTGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGACCAGGGCTACACACGTGCTACAATGGCGCATACAAAGAGAAGCGACCTCGCGAGAGCAAGCGGACCTCATAAAGTGCGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGTGGATCAGAATGCCACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCAAAAGAAGTAGGTAGCTTAACCTTCGGGAGGGCGCTTACCACTTTGTGATTCATGACTGGGGTG
U1D_3
AGCCCCAGTTACCACTTTCGCCCTAGGCCGCTCCTCTCGGTCACGGACTTCAGGCGCCCGCGGCTTCCATGGCTTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGCCATGGCTGATGCGCGATTACTAGCGAATCCAACTTCGTGGAGTCGGGTTGCAGACTCCAGTCCGAACTGGGACGGCTTTTCGAGATCCGCATCCCGTCGCCGGGTAGCCTCCCTCTGTGGCCGCCATTGTAACACGTGTGTCGCCCGGACGTAAGGGCCGTGCTGATTTGACGTCATCCCCCCCCTCCTCACACCTTGCGGTGGCAGTCCCGCCAGAGTGCCCAGCTTCACCTGATGGCAACTGGCGGCAGGGTTGCGCTCGTTATGGCACTTGAGCCGACACCTCACGGCACGAGCTGACGACAACCATGCAGCACCTCGGCACCTGTCCGAAGACCCACCCGTCTCTGGGTGGTTCAGGCGCCGTTCGAGCCCGGGTAAGGTTCCTCGCGTATCATCGAATTAAACCACATGTTCCTCCGCTTGTGCGGGCCCCGTCAATTCCTTTGAGTTTCATCGTTGCCGACGTACTCCCCAGGTGGATGGCTTATCGCTTTCGCTTGGCCACCGACAGTGTGTCGCCGGCGGTTAGCCATCATCGTTTACGGCGTGGAATACCAGGGTATCTAATCCTGTTCTATCCCCACGCTTTCGTGCCTCAGCGTCAGTTACGGATTCGCCAGATGCCTTCGCAATCGGTGTTCTGAGTGATATCTAAGCATTTCACCGCTACACCACTCATTCCTCCGGCGGCATCCGCACTCCAGCGCGACAGTATCAAGGGCAGCCCCGGAGTTGAGCCCCGGAATTTCACCCCTGACTTGACGCACAGCCTACGCACCCTTTAAACCCAATGAATCCGGATAACGCTCGCATCCCCCGTATTACCGCGGCTGCTGGCACGGAGTTAGCCGATGCTTATTCCCCTGGTACTCTCATCGGACGTGCGCGACGCCCTTATTGCCCCAGGCAAAAGCGGTTCACAGCCCATAGGGCCTCCTTCCCGCACGCGGCATGGCTGGTTCAGGCTTCCGCCCATTGACCAATATTCCTCACTGCTGCCTCCCGTAGGAGTATGGACCGTGTCTCAGTTCCATTGTGGGGGACCTTCCTCTCAGAACCCTACCGATCGTAGCCTTGGTGGGCCGTTACCCCGCCAACAAGCTAATCGGACGCGAGCCAATCCGTCGCCGCCGTAACTTTCAACAGAGACCCATGAGGGCCTCCGTCCCATCGGGGATTAGTCGGCGTTTCCACCGGTTGTCCCCGGGCAACGGGCATGTCGCTCACGCGTTACGCACCCTTCCGCCGGTCGCCGCCAGGACGTTGCCGCCCCGCGCTGCCCTCGACTTGCATGTGTTAAGCCTGCCGCTAGCGTTCATC

Thanks for your time!

colinbrislawn · December 5, 2019, 2:39pm

Good morning @binzuo

Welcome to the forums!

I have some bad news for you; long PacBio reads are supported by dada2 1.10, but they are not yet supported by the Qiime2 plugin.

But we are working on it! You can keep track of our progress here:

github.com/qiime2/q2-dada2

Add long-read support

opened 09:17PM - 20 Sep 18 UTC

benjjneb

**Improvement Description** Add long-read support **Current Behavior** We'v…e added support for PacBio long-read amplicon sequencing to the devel version of the R package and it seems to work quite well. Preprint: High-throughput amplicon sequencing of the full-length 16S rRNA gene with single-nucleotide resolution. **Proposed Behavior** I think it will make sense to add this as a tech-specific ```denoise-pacbio``` command in the plugin. This is similar to the ```denoise-pyro``` approach already in the plugin, with the purpose of having a dedicated command being to automatically turn on the right flags and options for PacBio data rather than relying on the user to do so. There is a downside in the repetition of much of the code between the different ```denoise-[technology]``` commands. **References** 1. Preprint: [High-throughput amplicon sequencing of the full-length 16S rRNA gene with single-nucleotide resolution](https://www.biorxiv.org/content/early/2018/08/15/392332).

For now, you would have to process your data directly with dada2 using R.

Colin

binzuo · December 5, 2019, 5:17pm

Thanks, Colin.

I use conda list to check the version of dada2, and I got this:
bioconductor-dada2 1.10.0 r351hf484d3e_0 bioconda

Doesn’t this mean the dada2 in qiime2-2019.10 is the version 1.10? I am rookie in this linux and qiime2 environment, so forgive me if I am wrong.

colinbrislawn · December 5, 2019, 6:10pm

Yes, you have dada2 1.10.0 installed. So you could open up R and use the settings for long reads.

But even though dada2 can use PacBio long reads, the Qiime 2 plugin does not have the option for it. If you look at the documentation for the dada2 Qiime 2 plugin, there are options for single, paired, and pyro, but no option for long reads.

Hopefully a 4th option for long PacBio reads will be added in 2020, but until then you can do this analysis in R.

Colin

binzuo · December 5, 2019, 7:34pm

OK, I get it. Thanks for your detailed and patient explanation.

system · January 6, 2020, 1:34am

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