How do i should proceed with 16S rRNA amplicon sequencing data from Illumina MiSeq using QIIME pipeline?
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4.5 years ago
antonioggsousa ★ 2.0k

Hello!

I'm a newbie in bioinformatics and with QIIME pipeline.

I received 16S rRNA amplicon sequencing data from Illumina MiSeq. 16S rRNA amplicons were sequenced from both ends - paired end sequencing. Among the different folders that i received, from the core sequencing facility, it was a "raw" folder. This contain individual fastq files for each end (forward and reverse) and for each sample (9 in total). So, the fastq files that i received were already demultiplexed, without barcodes, but with primers.

My questions are:

(1) How do i start? By merging the files and then removing the primers or the opposite?

(2) How do i perform any script that requires the "map.file" if i don't know the barcode sequences?

(3) How do i can perform downstream analysis, such as beta and alpha diversity, without map.file?

I started by running the multiple_join_paired_ends.py script. Now i'll try merge all the samples (9) in just one fastq file. Then i was thinking to remove both primer sequences, forward and reverse, through extract_barcode.py using the following argument --input_type -barcode_paired_end.

Please, help me. This is really frustrating for someone that just started to learn about bioinformatics and pipelines like QIIME without previous experience on that. Regards, @renh@

next-gen • 3.7k views
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Entering edit mode
4.5 years ago

(1) How do i start? By merging the files and then removing the primers or the opposite?

Ans: The first few steps would be:

A. Stitching HQ reads using FLASH or BBMerge

FLASH - https://ccb.jhu.edu/software/FLASH/

BBMERGE- http://jgi.doe.gov/data-and-tools/bbtools/bb-tools-user-guide/bbmerge-guide/

B. Removing chimeric DNA

C. Creating a mapping file.

Start here:

a. http://www.wernerlab.org/teaching/qiime [Tutorial with explanation]

b. [Youtube videos - extensive explanation]

c. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3249058/ [Research paper]

(2) How do i perform any script that requires the "map.file" if i don't know the barcode sequences?

Ans: See this link: http://qiime.org/documentation/file_formats.html

See similar links on QIIME1 google group forum:

https://groups.google.com/forum/#!topic/qiime-forum/Lo5DqFdfZh4

https://groups.google.com/forum/#!msg/qiime-forum/XDQnB_QPfHI/xOejWhxwiJwJ

Still, confused, post question on QIIME1 google group

Link: https://groups.google.com/forum/#!forum/qiime-forum

(3) How do i can perform downstream analysis, such as beta and alpha diversity, without map.file?

Mapping file will be required for few steps. Hence, learn how to create mapping file for samples without barcodes. See point (2) as mentioned above.

Try to create a file and run the validate_mapping_file.py script. It generates logs and error that are intuitive. Even, sometimes it generated "corrected" file for you.

Go step by step, it's very very easy.

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I'll follow step by step.

Thank you very much Vijay Lakhujani.

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