I am working with very few samples (4+4 samples in two groups) for microbiome analysis. The reads are limited around 50-1300 in those samples. While running dada2, I got vary limited number sequences are passing (after filtering, merged, non-chimeric steps). In two samples, I got 0 (zero) non-non …

Hi @dasqiime22 , [image] dasqiime22: around 50-1300 in those samples. This is already a very small # of reads to begin with, in most cases after processing, filtering, such low samples are removed. Here you are starting with these. Any particular reason why there are so few to begin with? …

Thanks for quick reply! Actually, I got the sequence (Multiplexed,paired end:R1,R2 and Barcode fastq files) from Argone LAB with many samples. But, I have to focus on few samples (8) only. So, I start with qiime2 (EMP protocol) to get demux (sample wise fastq files). Then I separate out the target…

Furthermore, I see the plot quality are moderately good in both 5 prime AND 3 end (>25-30). I check with no trim and also few trim at begining. qiime dada2 denoise-paired ** --i-demultiplexed-seqs demux-paired-end.qza ** --p-trim-left-f 6 ** --p-trim-left-r 6 ** --p-trunc-len-f 150 ** --p-trun…

Hi @dasqiime22 , Unfortunately these are extremely low sequenced samples and there isn't really anything meaningful you can do even if we were to fine-tune DADA2 parameters to retain more reads. Unless these are all negative control samples where you are not expecting any diversity, but I don't thin…

May be the de-multiplexing is the problem. The fastq files are extremely big (>2GB), I am not able to open it. But total I am getting only 45K reads altogether for all samples, which are very less I think. I received the three .fastq files: R1, R2, barcodes. Convert the files into .fastq.gz (as …

Hi @dasqiime22 , 45K reads across 8 samples is certainly much more normal and totally workable. I think the hunch that you are losing your reads at the demultiplexing step might be right after all. Some things to check, are you sure your data is actually in EMP format ? This format is very specific a…

Many many thanks! No. I got 45K for all samples, and 3.2K for my 8 samples which is not sufficient. My data is multiplexed, paired end and barcodes is in separate file, altogether three files (R1,R2, I1), so, I think I have to use EMP paird end protocol. Is it write? Today, I also try EMP and go…

Hi @dasqiime22 , [image] dasqiime22: No. I got 45K for all samples, and 3.2K for my 8 samples which is not sufficient. Oops, sorry I thought you meant 45k across your 8 samples. If you indeed have confirmed ~3.2k reads for you 8 samples before demultiplexing then it it would seem your origina…

Thanks 100000k! Probably, My problem is solved. I see in the original files, I have around 2 corers sequences for all samples. Just now I have executed with EMP and --p-no-golay-error-correction, without any --p-rev-comp-barcodes **** or –p-rev-comp-mapping-barcodes **, and I got total 994891 (10…

Another thing you can try to increase the ASVs detected in low read/highly multiplexed samples is to process the full dataset (or at least more of the samples) through dada2 before selecting only the 4 samples you care about. The dada2 algorithm uses lower-abundant sequences to distinguish errors f…

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