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Do Any Next-Generation Sequencing Platforms Generate Reads With Ambiguous Bases (Other Than N)?

I've never seen them in our own Illumina data, but I don't know if that's because the platform (including software) doesn't generate them, or if the users of the GA Pipeline in our core facilities selected certain parameters to suppress them.

next-gen sequencing
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updated by Aaron Statham Aaron Statham • written by Bio_X2Y Bio_X2Y

4 answers

I dont think so.. Even I never say them our Illumina reads. Sequencer's algorithm are tuned in a way that if the base signal is poor or ambigous then report it as N or only one with higher signal.

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written by Rm Rm

Many algorithms will only use the nucleotide with the highest probability and ‘call’ that location in the read, ignoring the other three probabilities.

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written by Rm Rm

Thanks for the link, it looks useful

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written by Bio_X2Y Bio_X2Y

For illumina, the intensity text files have four scores per base call (_int.txt) in this format: [?]t{%5.1f %5.1f %5.1f %5.1f}+ with value range [ -16384.0,16383.0]. you can use it to decide whether ot not ambiguous base

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written by Rm Rm

Cheers for that, it ties in with Daniel's answer.

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written by Bio_X2Y Bio_X2Y

I don't think it would be useful as a correct coverage under a given position will quickly solve the ambiguity.

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written by Pierre Lindenbaum Pierre Lindenbaum

I'm not 100% sure, but I do know that Illumina does create raw files at some point in the processing pipeline that have four probabilities at each position: 1 for each nucleotide. At some point in the pipeline, however, one of the 4 nucleotides is chosen as the most probable and its corresponding quality value is the one you see downstream (in, say, a Fastq file). I'm not sure how ambiguities are resolved if two nucleotides have nearly identical probabilities.

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written by Daniel Standage Daniel Standage

thanks for the background info

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written by Bio_X2Y Bio_X2Y

The Illumina pipeline did at some point create .prb files, which had the probability for each of the 4 nucleotides. Some aligners take .prb files as input (eg slider using the extra information to try to make better SNP calls. I never really saw the point though!

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written by Aaron Statham Aaron Statham

GNUMAP algorithm also does it similarly

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written by Rm Rm

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