4D Nucleome Network Wiki

Agenda

1. Presentation by Peter Fraser, single cell HI-C method. \(protocol attached). - Update on sub working groups: - PLAC-seq/HiChIP: \(Miao Yu)
   1. single cell Hi-C (Burak)
   2. other sub working groups

Single cell Hi-C protocol (Takashi Nagano and Peter Fraser)

Presentation is uploaded on 4DN Wiki at the following link: https://docs.google.com/a/4dnucleome.org/viewer?a=v&pid=sites&srcid=NGRudWNsZW9tZS5vcmd8NGQtbnVjbGVvbWUtd2lraXxneDozZjUyMDBkZTk0NTZjZmI4)

Presentation’s Questions

1.- What are the QC measures that indicate that the single cell Hi-C method was a success?

1. Digestion efficiency by PCR and ligation junctions cut sites. 60% digestion was obtained and was good.
2. The bioanalyzer trace is the first QC. It show a broad peak with a low primer-dimer formation.

2.- Will these QC metrics be included as metadata to indicate the quality of the experiment? Raw data need to be submitted as well as barcodes. Some other experimental metadata should also be included (picture from Bioanalyzer, for example)? Dr. Fraser will discuss with Takashi on including bioanalyzer figures, tests, etc.

Takashi shall discuss with Burak about implementation. 6% trans contacts appears to be impressive for contact profile.

Fraser Lab has a cut off to filter cells with not enough number of reads. Yaniv had a presentation about this filtering (the presentation is at Single-cell OMICS sub-working group meeting and is available on the wiki at the following link: https://docs.google.com/viewer?a=v&pid=sites&srcid=NGRudWNsZW9tZS5vcmd8NGQtbnVjbGVvbWUtd2lraXxneDoyMTcyOWE4NTFiNDBlMDcw).

3.- Have you look at the short range to long range ratio what proportion of the reads are from contacts of less than 20kb?

In a single cell Hi-C experiment you get exactly the same contact probability curve as for a population Hi-C experiment. For example, single cells have different profile depending on they are in the cell cycle. For example, mitotic cells have enrichment in 4-12 megabases, cells that are coming out from mitosis have very long range contacts that are from 50-100 megabases and as the cells enter cell cycle have an increase in contacts to two megabases, so it's constantly changing, thus the population is made of different contact profiles.

4.- The QC metrics is a pool of single cells,you also have metrics that state that the quality of the data came from single cell?

Yes, Janine, presented the criteria used for the single cell metrics. We need to link Janine presentation with this presentation. \ 5.- Bimodal distribution of some cells shows fewer contacts per cell and other have more, is the blue line intended for a cut-off between the two distributions?
The actual reason of the bimodal distribution is not known yet and the blue dashed line is the mean contact number per cell and is not intended to be a cut-off for the two models. The cut off was set of for 10,000 read pairs. 6.- Minimum standards thresholds for this dataset?
Two million reads per cell (average) is the minimum and is not giving saturation (10-14 million read pairs gaves saturation). However, this protocol is not like the 3C version that requires to sequence much more, such as 20 million reads per cell where you have to sequence the whole genome to find restriction sites of the ligation products if you have biotinylated ligation junctions. Thus, we are going for maximum coverage not for maximum high throughput. 7.- Minimum number of cells?
Depend of the experiment, obviously 10 cell do not help. Hundreds or thousands of cell is much better. 96 cells are recommended for general experimental purposes. 8.- Shall we have a QC to indicate that the home made TN5 perform as well as Mgr1? \Currently, this has been testing. It works fine in a population Hi-C experiment, but not in single cell experiment in house growing TN5, however we are getting a high percentage of GC rich read pairs that are unmappable to the human genome, so we are assuming that this is bacterial DNA contaminating the prep. We are looking to remove this without destroying the activity of the enzyme and also not preventing downstream steps. If the prep can be cleaned up then much of the cost can be saved as well.

Current status in sub working groups

At the moment the single cell OMICS sub-working group is on hold trying to collect data. Waiting for data submission and starting working with Leonid group.

Miao Yu on PLAC-Seq protocol: the updates will be sent to the working group via email.

OMICS WG is trying to encourage labs to sign up for additional protocols. The working group will continue to get this going.