CUTTAG duplication

[lengfei5]

Hi there,

Thank you for the CUT & TAG tutorial which is extremely helpful.
I have a question regarding the duplication removal in the Cuttag samples.
I read that in the tutorial
''
CUT&Tag integrates adapters into DNA in the vicinity of the antibody-tethered pA-Tn5, and the exact sites of integration are affected by the accessibility of surrounding DNA. For this reason fragments that share exact starting and ending positions are expected to be common, and such ‘duplicates’ may not be due to duplication during PCR. In practice, we have found that the apparent duplication rate is low for high quality CUT&Tag datasets, and even the apparent ‘duplicate’ fragments are likely to be true fragments. Thus, we do not recommend removing the duplicates. In experiments with very small amounts of material or where PCR duplication is suspected, duplicates can be removed.
''
I obtained samples with 70-90% duplication, and I am just wondering how to make a decision if those duplication are due to PCR or not.

Many thanks.

Best,
Jingkui

[yezhengSTAT]

Hello Jingkui,
Such high duplication rate is a sign for duplicates removal. What is your sequencing depth? Did you over-sequence the samples? The comments and questions next to step 12.2 at https://www.protocols.io/view/cut-amp-tag-data-processing-and-analysis-tutorial-bjk2kkye maybe helpful.

Best,
Ye

[lengfei5]

Hi Ye,

Thank you for your quick answer.
You are right that we probably over-sequenced the samples.
We sequence > 100M for H3K27ac and H3K4me1, because those samples from axolotl with 10x bigger genome than human.
Previous test with 50M sequencing depth have very low signal-to-noise ratio.
I will remove duplication for our samples.

Best,
Jingkui