Nextera Transposon Contamination
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6.9 years ago
Daniel E Cook ▴ 240

Has anyone dealt with Nextera transposase sequence contamination before? Wondering if this is something we should work to eliminate. What would be an acceptable level of contamination?

Thank you - 

nextera fastq • 11k views
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Hi all,

I've just found in Nextera™ DNA Sample Prep Kit (Illumina®-Compatible)

Transposon End Sequence: The 19-bp transposon DNA sequence is present at the 5 ′ end of all Illumina®-compatible libraries. However, the 19-bp transposon DNA sequence is NOT sequenced on the Illumina® platform. The Nextera Read 1 and Read 2 primers anneal to this sequence so that the first nucleotide sequenced is target DNA.

Looks like It is not true that this sequence is NOT sequence if we have it in our reads... according to this paper it shouldn't?

Does this could be cased by library contamination?

Best,

Agata

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It's not sequenced if your inserts aren't shorter than the read length. Essentially, you only sequence it if you read through the insert in the other adapter. Which matches the observations here: the adapter is reached at the ends of the reads.

What the text you copied means it that it isn't sequenced at the beginning of the read.

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6.9 years ago
Adrian Pelin ★ 2.5k

I just had a sample where 90% of the output was this crap... Even so, after removing it, the data was similar to another technical replicate that did not have the transposase, so I think your levels are usable.

 

What I used is SeqPrep, so:

SeqPrep -A GTGTATAAGAGACAG -B CTGTCTCTTATACAC -f in_r1.fastq -r in_r2.fastq -1 out_noTP_r1.fastq.gz -2 out_noTP_r2.fastq.gz
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I do not know where you got these two sequences (-A and -B) from, but for Nextera, you have to trim CTGTCTCTTATA for both the forward and reverse read. You can have a look here at the Illumina Adapter Guide for the Nextera adapter sequence, if you want to check it in detail.

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The part I have in -B is the same sequence you have indicated, just longer by 3 bp. The part I have in -A is just the reverse complement of -B. This was 2 years ago so I do not remember 100% where I found it, but I remember validating it visually on misalignments.

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The link is unavailable. Do you have another?

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here is the link Illumina Adapter Sequences

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That link is now also dead, you may like this one.

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4.4 years ago
aln ▴ 290

Have the same problem, used trim_galore with --nextera flag, which seemed to help. Also here is relevant post by Nick Loman, but unfortunately, the pictures are missing.

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