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Whole genome sequencing, different sequencing platforms, different quality control?

Hi there,

I am working with bacteria whole-genome sequencing data and planning to search SNPs.

However, I have collected the public whole-genome sequencing raw read data from NCBI. Different samples are sequenced with different sequencing platforms, such as Illumina Miseq, Illumina Novaseq, Illumina Hiseq.

While read quality control, trimming adapter sequences and low quality reads, how should I consider different samples from different sequencing platforms? For example, different minimum length.

In addition, while mapping reads to the reference genome and calling genomic variants, should I also give different thresholds?

Thanks in advance for your answers.

wgs snp bacteria

3 answers

Different samples are sequenced with different sequencing platforms, such as Illumina Miseq, Illumina Novaseq, Illumina Hiseq.

These are all Illumina sequencers so the basic technology is identical (though the chemistry may be different). You should be able to use standard Illumina QC and analysis techniques for all of these data.

I have collected the public whole-genome sequencing raw read data from NCBI.

and

I am working with bacteria whole-genome sequencing data and planning to search SNPs.

Collecting public sequence data and trying to do meta-analysis to identify SNP's sounds vague. What do you actually hope to get from this? Are you focusing on sequence from a particular species/strain?

Thank you for your kind answers.

I am focusing on sequence from a particular species, and comparing between strains where they have genomic variants that may change their phenotype.

I have some additional questions about standard Illumina QC.

I understand that the Illumina sequencers share the basic technology, but the average read lengths differ, ranging from 75bp to 300bp.

Then, should I set different minimum length? Because 50bp from the sample of average read length 75bp and 300bp should imply different meanings. Previous sample wouldn't be a big problem, but the latter one would mean severe quality problem, doesn't it?

The simplest thing to do to harmonize all your different reads is to trim them all to the same length. You want the tricky repetitive regions to be equally difficult in all samples.

The chemistry differences between the different platforms are very small, so I wouldn't worry about that.

Thank you for your nice answer.

I have some additional questions about the repetitive regions you mentioned.

The average read lengths differ from 75bp to 300bp. If I trim them all to the same length, then I think there will be a significant loss of information. Rather then trimming them to the same read length, how about masking the repetitive regions of the reference genome?

Then, should I set different minimum length? Because 50bp from the sample of average read length 75bp and 300bp should imply different meanings. Previous sample wouldn't be a big problem, but the latter one would mean severe quality problem, doesn't it?

In this case, I would suggest that you set a standard on what read-Lenght you want to consider. say you set 150bpx2 as minimum, then you can put all the data through 1 standard pipeline, with similar flags for each tool. This will be helpfull in dealing with batch-effect (if that can happen in WGS data too)

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