Diverse logics and grammar encode notochord enhancers

This GitHub repository contains the code used in the analyses for the following manuscript:

Song, Benjamin P+, Michelle F. Ragsac+, Krissie Tellez, Granton A Jindal, Jessica L Grudzien, Sophia H Le, Emma K Farley. "Diverse logics and grammar encode notochord enhancers." In submission, 2022.

• These authors contributed equally

https://www.biorxiv.org/content/10.1101/2022.07.25.501440v1

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Background Information

For this study, we searched through the Ciona intestinalis genome to find regions containing both Zic and Ets binding sites that could confer notochord activity in the developing embryo. To do this, we first searched through the genome for candidate regions to include within a massively-parallel reporter assay (MPRA) or "enhancer screen."

This repository contains analyses for the genome search (01_genome-search/) and the MPRA performed in whole Ciona embryos (03_bulk-screen). We call the linking between barcode tags and associated enhancer the "dictionary" analysis (02_enhancer-dictionary). If you would like to perform the analyses for the whole embryo MPRA on your own computer, you can find the raw sequencing data under the following SRA ascession identifier: PRJNA861319.

Additional Data Resources

Within this study, we also used additional data resources provided by the scientific community in our analyses:

Resource Name	Resource Usage	Resource Link
Ciona intestinalis Genome Sequence (JoinedScaffold.fa)	Genome Search	Ghost Database
Ets1 Binding Data for Mus musculus (Ets1/Ets1_8mers.txt)	Genome Search	UniPROBE Database

Script Parameters

For this study, we used several scripts to analyze our data. We wanted to share the parameters that we used for each of the software tools:

Software Tool / Script Name	User-Defined Parameters
genome-search.py (Custom Script)	Window Size = 30 bp
FLASH Software Tool	Maximum Mismatch Density = 0.0

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Analysis Procedure

1. Perform the genome search within Ciona intestinalis for regions with at least one Zic site and two Ets sites using 01_genome-search/genome-search.py with a flanking window size of 30 bp.
2. Select regions from the search to include in this study.
3. Assemble the barcode tag-enhancer sequence dictionary and analyze the sequencing data:
   1. Determine the sequencing quality with fastqc (02_enhancer-dictionary/01_fastqc).
   2. Because we have overlapping sequence between our reads, assemble the entire read sequence using FLASH (02_enhancer-dictionary/02_flash).
   3. Consolidate unique reads by "collapsing" them and tallying up the number of times a unique sequence appears in our data (02_enhancer-dictionary/03_collapse).
   4. Analyze and determine the barcode tag-enhancer sequence dictionary associations (02_enhancer-dictionary/04_assemble).
4. Analyze the bulk, whole-embryo massively-parallel reporter assay (MPRA) in Ciona intestinalis:
   1. Determine the sequencing quality with fastqc (03_bulk-screen/01_fastqc).
   2. Consolidate unique reads by "collapsing" them and tallying up the number of times a unique sequence appears in our data while also applying a read count filter (03_bulk-screen/02_collapse/filter-collapse.py with outputs in 03_bulk-screen/03_assemble/filtered-inputs/).
   3. Analyze the library and determine the distribution of activities for our enhancers (03_bulk-screen/03_assemble/).