==== Omics Data Standards WG - Minutes 01-22-2018 ====

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===== Agenda =====

  - Update from sub-working group on PLAC-seq/HiChIP (Miao Yu)
  - Update from sub-working group on single cell Hi-C (Burak Alver)
  - Presentation of SPRITE (Sofia Quinodoz/Guttman Lab) followed by discussion.

===== Update from sub-working group on PLAC-seq/HiChIP (Miao Yu) =====

Preprocessing is similar to HiC. Difference between PLAC-seq and HiChIP is minimal, mainly in how the libraries are generated. It’s currently very hard to compare since the cell types are different. Therefore, we would need to generate data from the same cell type and stage.

Yijun Ruan’s lab has a working protocol for in situ ChIA-PET, by introducing in situ digestion and ligation in the protocol and would like to join the sub-working group, and share the protocol in two weeks for comparison.

===== Update from sub-working group on single cell Hi-C (Burak Alver) =====

Yaniv Rubin presented their data-processing pipeline and QC metrics. DCIC has decided that different protocols from the labs for single cell Hi-C are different enough. Therefore, they won’t pursue a uniform single cell Hi-C protocol. However, a uniform data standard and analysis pipeline is still favorable and DCIC is currently requesting data from the participating groups in order to form such a pipeline and standard.

===== Presentation of SPRITE (Sofia Quinodoz/Guttman Lab) followed by discussion =====

SPRITE protocols can be downloaded here: [[https://drive.google.com/file/d/17hkhfodJ-XnRME38hWfPGWXHAP69IcyR/view|https://drive.google.com/file/d/17hkhfodJ-XnRME38hWfPGWXHAP69IcyR/view]]

[[https://drive.google.com/file/d/1sUsjuoG4ohIs5Aj7yMk_zoBrUZx4tadz/view?usp=drive_web|Presentation could be found here: 20180121-Omics-SPRITE-DNA.pptx]]

Sequencing barcoding can be used to tag interacting RNAs & DNAs by separating complexes in different wells and tag with different barcodes. Although different complexes may end up in one well and get the same barcode in one round, by increasing rounds for pooling-tagging process (5 for example), they will be marked uniquely.

Protocol: crosslink nuclei -> fragmentation with sonication -> couple to NHS-activated magnetic beads -> split-pool tagging (x5) -> library preparation and sequence

All adaptors have 5’ sticky ends, are 5’phos modified and have unique barcodes. Illumina read 1 sequence is included in the first barcode (DPM). Barcode are grouped into “odd” and “even” so that same barcode won’t be consecutively ligated. DPM also have a 3’spacer so during library construction only one side of barcode will be included.

Cross-species library construction is used to analysis the amount of spurious ligation. When molecule-to-bead ratio is high, cross-species ligation rate is high. This is used to choose the proper molecule-to-bead ratio.

DPM ligation and barcode ligation products can be ProK eluted to get QCed via bioanalyzer.

Taking 5% aliquot of beads with 10-12 cycles of PCR amplification to determine the number of uniquely-tagged molecules, then elite many 1-5% aliquots to achieve a 1.5x - 2x coverage. This is needed to sample the majority of tagged molecules, thus capturing most interactions.

Post-sequencing QC including ligation efficiency (by counting number of barcodes for each read), how many reads are sharing one barcode (used to optimize fragmentation process), if all molecules tagged are sampled (observed vs. estimated distinct molecules based on the number of reads).

SPRITE pipeline can be seen at GitHub: [[https://www.google.com/url?q=https://github.com/GuttmanLab/sprite-pipeline/wiki&sa=D&sntz=1&usg=AFQjCNHJY4EXAMUILHP5cFW5VWa60KTD3w|https://github.com/GuttmanLab/sprite-pipeline/wiki]]

Models of the devices used in the protocol can also be included, such as model of the sonicator.

Comments can be sent to Bing Ren, Sofia Quinodoz or the working group in general for discussion within the 30-day period. Guttman Group would also like to help anyone interested in the protocol.
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