Multi-way Hub Identification

Last updated: June. 3, 2024

Prerequisites

Packages

• R (tested on 4.3.1)
  • Pairtools (tested on 1.0.2)
• (other packages required as dependencies)

Data

• Sam/Bam files for each single-cell in a directory

Installation

Download the github repository. Install all the requirements.

Test Run

1. Make sure the example data (LC716 dataset) is downloaded.

2. Change reference genome dependency files path location in the Rscript "Multiway.Contacts.Step1.R" , "Hub.Step2.R" . We have provided files for mm10 i.e mm10.blacklist.bed , mm10.chrom.sizes , F_GC_M_MboI_10Kb_el.mm10.txt , 10KbBin_mm10_blacklist.txt.

Change the path of above mentioned files in following variables in R script "Multiway.Contacts.Step1.R" :

mm10.chrom.size = "./mm10.chrom.sizes"   ######## Path to mm10 chrom sizes file ########
mm10.blacklist.region = "./mm10.blacklist.bed" ####### Path to mm10 Black list regions #########

Similarly change the path of above mentioned files in following variables in R script "Hub.Step2.R" :

mm10.blacklist = '10KbBin_mm10_blacklist.txt'     ##### Path to mm10 Blacklist region
annotation = 'F_GC_M_MboI_10Kb_el.mm10.txt'       ##### Path to mm10 10Kb MboI annotation file

3. Set following two path for dataset in script "Multiway.Contacts.Step1.R" :

dir1 = "./example.data/bam.sc/"      #### Path to bam/sam files ######
dir2 = "./example.data/chimeric.sc.pairs.mapq40/"     #### Path to save all intermediate files ######****

Set following path for dataset in script "Hub.Step2.R". We have provided example for "cellnames" , "color.loc" in example.data directory. :

dir<-'./example.data/chimeric.sc.pairs.mapq40/'   #### Path to directory where all intermediate result files were saved in step1 (dir2 in step1)

cellnames = "./example.data/LC716_MAPQ40_cell_names_Shreya_light.txt"     ###### Path to example data cellnames file
color.loc = './example.data/color_20_YangOrder.txt'                       ###### Path to Color assigned to each celltype 

metadata = './052124_LC716_MAPQ40_metadata_MultiContact.txt'      ###### Filename to save multiway Contacts metadata file
final.result = './hub_20celltypes_MAPQ40.txt'                     ###### Filename to save final hub file

4. Note : Unzip all sam files in folder "./example.data/bam.sc/" before running further commands.

5. Run command below :

Rscript Multiway.Contacts.Step1.R

Then run second step when first step is complete :

Rscript Hub.Step2.R

2. File format requirements

2.1 Sam file

> head CTTCTAAAGAACTAAC.OGC.int.sam
@SQ	SN:chr1	LN:195471971
@SQ	SN:chr2	LN:182113224
@SQ	SN:chr3	LN:160039680
@SQ	SN:chr4	LN:156508116
@SQ	SN:chr5	LN:151834684
@SQ	SN:chr6	LN:149736546
@SQ	SN:chr7	LN:145441459
@SQ	SN:chr8	LN:129401213
@SQ	SN:chr9	LN:124595110
@SQ	SN:chr10	LN:130694993
@SQ	SN:chr11	LN:122082543
@SQ	SN:chr12	LN:120129022
@SQ	SN:chr13	LN:120421639
@SQ	SN:chr14	LN:124902244
@SQ	SN:chr15	LN:104043685
@SQ	SN:chr16	LN:98207768
@SQ	SN:chr17	LN:94987271
@SQ	SN:chr18	LN:90702639
@SQ	SN:chr19	LN:61431566
@SQ	SN:chrX	LN:171031299
@SQ	SN:chrY	LN:91744698
@PG	ID:bwa	PN:bwa	VN:0.7.17-r1188	CL:bwa mem -SP5M -T0 -t16 /projects/ps-renlab/share/bwa_indices/mm10.fa /home/y2xie/scratch/62.schiC_MouseCortex_NovaSeq_230723/02.trimmed/.///LC716_R1_BC_cov_val_1.fq.gz /home/y2xie/scratch/62.schiC_MouseCortex_NovaSeq_230723/02.trimmed/.///LC716_R3_BC_cov_val_2.fq.gz
CTTCTAAAGAACTAAC:A01535:363:HFY77DSX7:4:1101:28004:1110	65	chr11	63157895	60	151M	=	63144151	-13745	TGTCTGTACAGAGGTTTGGGGCTTCACCAGCATATCTGCACCAGGTTGCTTCATAGAAGAGTTAGGCAGGCTCATCTTGCAGCCTGGGTCCTCACTGCCTGTCTTGAATGACCTAGGAAGAAGTGTTATGCTGAAAGAATGAGAAATGAGG	:F:::FFFFFFFFF:FF::FFFFFFFFFFFFFFFF,FFFF:,:FFF:F:FFFFFFF,FFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFF:FFF:FFFFFFFFFFFFFFFF:FF:F,FFFFFFFFF,F:F:FFF:FFFFFFFFF	NM:i:0	MD:Z:151	MC:Z:84M66S	AS:i:151	XS:i:0
CTTCTAAAGAACTAAC:A01535:363:HFY77DSX7:4:1101:28004:1110	129	chr11	63144151	60	84M66S	=	63157895	13745	CTATTGTGCTCACTAGCTTCTTATGGTGCTAGTGCAAAAGTGGGCACACTCTAGCCTTAGGGGCCAAAGGAAAGACACCACTTGTTCTAGGCGGAGGTGGGCATTTACAACACTAGGCAGGGACGATGTGTCTGAGCCTCCCAGCTCAGA	F,,,:FFFFFF:FF:F,,,F,:,,FFFFF:,FFF,FF,,FFFFF:FFF:FF:,F::,F,FF:F:F:,,FF,,:F,F,F,,,::FFFFF,FF:FF,FFFFFFF,,,,F,:,FF::,FFF:FFF:,FF,F,FFFFFFFFF:FFF,FF,F:FF	NM:i:1	MD:Z:46A37	MC:Z:151M	AS:i:79	XS:i:19	SA:Z:chr11,63158153,-,65M85S,60,1;

2.2 Metadata file with columns "Cellname","Celltype"

> head LC716_MAPQ40_cell_names_Shreya_light.txt
AAACGAACAATCAGGG ITL6GL
AAACGAACATCCGTGG D12MSN
AAACGAAGTTAGGCTT ITL23GL
AAACGAATCTCAGATG CTGL
AAACTCGAGGACTAGC MGL
AAACTCGAGTCTGCTA CTGL
AAACTCGAGTGAATAC ITL45GL
AAACTCGCACTTTGGA MGL
AAACTCGCATAAAGTG ITL5GL
AAACTCGCATCCATAG ASC

2.2 Color assignment file with columns "Celltype","Color"

Subclass	Subclass.color
ASC	#A13F9E
OGC	#985695
OPC	#B9A0D6
MGL	#DF8DB1
VLMC	#A78BDF
ITL23GL	#E59D9F
ITL45GL	#DDA58F
ITL5GL	#E1A462
ITL6GL	#9E4954

Contact Us

For any question, contact Ming Hu (hum@ccf.org), Shreya Mishra (mishras10@ccf.org), or submit an issue on GitHub.