Hi everyone,
I would like to get into contact with researchers that are familiar with analyses of data obtained with Nextera Miseq Illumina. Basically, I am having problems in merging and/or assembling the paired-end reads that I have obtained from an Illumina run on a PCR fragment from obtained using the AMF primers AML1-AML2. This fragment was used to construct a Nextera XT DNA Library before sequencing. I am using CLC genomics and the protocols that I have seen refer to the analyses of amplicons directly, and not from a DNA that has been first fragmented and tagged with sequencing adapters.
Any tips would be welcome.
Thanks to all!
Both QIIME and mother offers solution for 18S data analysis.
You may also consider PEAR for merging
http://sco.h-its.org/exelixis/web/software/pear/
I have tried the FLASH (http://ccb.jhu.edu/software/FLASH/) to merge the pair-end sequence and the further analysis mainly based on the Qiime
If you are using CLC genomics, one thing to try out will be to attempt to assemble the reads into fragments. You could merge paired reads, but that decision depends on the average library size you were dealing with. Also, CLC workbench does allow assembly using paired reads. Remember to feed in the max fragment size (you probably know the length of the fragment you amplified) when assembling to reduce any assembly artifacts.
The assembled (hopefully full length or almost full length 18S)contigs can then be run through QIIME, Mothur or any pipeline of your choice. The trickier thing would be to adjust the otu table with counts of reads mapping to each contig (which map to the reference sequence). One paper that I came across that used a similar approach is Miller et al (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3566076/). Though they targeted the 16S fragment, the approach is probably relevant.
All the best !
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