Hi,
I have biological replicates: they come from the same raw sample (tube of sediment) but from different DNA extraction & sequencing experiments. These are metagenomes and I used whole genome sequencing.
If I treat them separately, the abundances are consistent, some of them cluster together on a PCoA but not so well on hclust.
Question:
1) Should I concatenate the raw fastq files together beforehand?
2) Or should I treat them separately until after the alignment to a db step and then sum the counts of each replicates together?
What is the best procedure?
Thanks
Biological replicates of environmental samples will have some variations. Additionally, the quality/quantity of the DNA, as well as the library construction method will also interfere with the sequencing outcome.
I'm assuming you extracted DNA from the biological sample using the same method and at the same time. In addition, both were sequenced using the same library preparation techniques.
The final choice to merge or not will depend on the aim of the experiment. For comparative analysis, you can merge the samples to get a single assembly and then get the gene/contig counts from non-merged biological replicate reads. It is recommended to get the count data from both the biological replicates (non-merged) as you will be comparing it with other metagenomic samples. Furthermore, for statistical analysis, you will need replicate samples
If the comparative analysis is not your aim, you can merge the biological replicates before or after, both won't matter. But it will influence your assembly. For this, it is better to use merged samples for assembly.
Note: If you are aiming for taxonomic and functional resolution at the strain level, then the co-assembly (by merging cleaned fastq files) approach is not recommended.
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