/ASE_Meta_Analysis

ASE_Meta_Analysis

Primary LanguageRGNU General Public License v3.0GPL-3.0

ASE Meta Analysis Paper

Code to accompany Edwards et al, 2023, https://doi.org/10.7554/eLife.83364

Citation

Edwards, C.A., Watkisnon, W.M.D., Telerman, S.B., Hulsmann, L.C., Hamilton, R.S. & Ferguson-Smith, A.C. (2023) Reassessment of weak parent-of-origin expression bias shows it rarely exists outside of known imprinted regions. eLife 12:e83364 [eLife] [DOI]

bioRxiv preprint available at: https://doi.org/10.1101/2022.08.21.504536

Department of Genetics, University of Cambridge, Downing Street, Cambridge, CB2 3EH

Methods

Raw sequencing read FASTQ files were downloaded from EMBL-EBI European Nucleotide Archive for each of the RNA-seq data sets ( [DOI] [DOI] [DOI] [DOI] [DOI] ). Low quality bases and adapters were removed with trim_galore (v0.4.1) [WEB]. SNPSplit (v0.3.4) [DOI] was used to separate reads by parent of origin, which first required the preparation of allele-specific reference genomes for C57BL6/CAST_Eij and CAST_Eij/FVB (based on C57BL6) with the following commands SNPsplit_genome_preparation --vcf_file mgp.v5.merged.snps_all.dbSNP142.vcf.gz --reference_genome GRCm38_fasta/ --strain CAST_EiJ and SNPsplit_genome_preparation --vcf_file mgp.v5.merged.snps_all.dbSNP142.vcf.gz --reference_genome GRCm38_fasta/ --strain CAST_EiJ --strain2 FVB_NJ --dual_hybrid. VCF files for strain specific SNPs were obtained from www.sanger.ac.uk/data/mouse-genomes-project. The Clusterflow pipeline tool was used to enable running multiple jobs in parallel across multiple processors on a HPC, however all scripts are also compatible with running on a single processor [DOI]. Trimmed reads were aligned to either the C57BL6/CAST_Eij or CAST_Eij/FVB reference genomes using HiSat2 (v2.1.0)[DOI], run via the hisat2 ClusterFlow module. Aligned reads were then name sorted to be compatible with SNPSplit, run via the samtools (v1.9)[DOI] Clusterflow module. Aligned files were run through SNPSplit to produce separated parent-specific alignment files using the SNP files produced by the genome preparation: all_SNPs_CAST_EiJ_GRCm38.txt.gz or all_FVB_NJ_SNPs_CAST_EiJ_reference.based_on_GRCm38.txt.gz. A custom Clusterflow module was created for SNPsplit [SNPSplit.cfmod]. Gene counts from each parent-specific alignment BAM file produced by SNPSplit were calculated using featureCounts (v1.5.0-p2)[DOI] via Clusterflow. A custom Rscript DESeq2_featureCounts_2_CountsTables.R is used to make a single counts table, including normalised reads, from the individual featureCount files. All scripts to reproduce the analysis are freely available at github.com/darogan/ASE_Meta_Analysis.

Analysis: Quality Check

A sanity check to make sure all the samples behave in a similar, or expected manner

Dataset Read Counts
Andergassen_2015
[PDF]
Babak_2015
[PDF]
Bonthuis_2015
DRN TO ADD
[PDF]
Perez_2015
[PDF]
Teichman_ESCs_NPCs
[PDF]

Data Sets

Dataset Publication Details
Andergassen_2015 [DOI] Cast/EiJ X FVB strains
Babak_2015 [DOI] C57Bl/6J x Cast (maternal allele first)
Bonthuis_2015 [DOI] C57Bl/6J x Cast (maternal allele first)
Perez_2015 [DOI] F1i (F1 hybrid) C57Bl/6J father and Cast/EiJ mother = CB ; F1r (F1 hybrid) Cast/EiJ father and C57Bl/6J mother = BC
Teichman_ESCs_NPCs [DOI] Cell: C57Bl/6J and Cast/EiJ (maternal allele first)

Andergassen_2015

Accession Tissue Cross Replicate
SRR3085966 adult Brain CF 1
SRR3085967 adult Brain CF 2
SRR3085968 adult Brain FC 1
SRR3085969 adult Brain FC 2
SRR3085970 adult Liver CF 1
SRR3085971 adult Liver CF 2
SRR3085972 adult Liver FC 1
SRR3085973 adult Liver FC 2
SRR3085990 adult Leg Muscle CF 1
SRR3085991 adult Leg Muscle CF 2
SRR3085992 adult Leg Muscle FC 1
SRR3085993 adult Leg Muscle FC 2

Babak_2015

Accession Tissue Cross Replicate
SRR823449 Muscle CB 1
SRR823450 Muscle BC 1
SRR823469 Liver BC 1
SRR823474 Liver CB 1
SRR823485 Hypothalamus CB 1
SRR823478 Hypothalamus BC 1
SRR823461 Cerebellum BC 1
SRR823458 Cerebellum CB 1
SRR823472 Adult Whole Brain CB 1
SRR823473 Adult Whole Brain BC 1

Bonthuis_2015

Accession Tissue Cross Replicate
SRR2086215 ARN BC 1
SRR2086216 ARN BC 2
SRR2086217 ARN BC 3
SRR2086218 ARN BC 4
SRR2086219 ARN BC 5
SRR2086220 ARN BC 6
SRR2086221 ARN BC 7
SRR2086222 ARN BC 8
SRR2086223 ARN BC 9
SRR2086224 ARN CB 1
SRR2086225 ARN CB 2
SRR2086226 ARN CB 3
SRR2086227 ARN CB 4
SRR2086228 ARN CB 5
SRR2086229 ARN CB 6
SRR2086230 ARN CB 7
SRR2086231 ARN CB 8
SRR2086232 ARN CB 9
SRR2086233 DRN BC 1
SRR2086234 DRN BC 2
SRR2086235 DRN BC 3
SRR2086236 DRN BC 4
SRR2086237 DRN BC 5
SRR2086238 DRN BC 6
SRR2086239 DRN BC 7
SRR2086240 DRN BC 8
SRR2086241 DRN BC 9
SRR2086242 DRN CB 1
SRR2086243 DRN CB 2
SRR2086244 DRN CB 3
SRR2086245 DRN CB 4
SRR2086246 DRN CB 5
SRR2086247 DRN CB 6
SRR2086248 DRN CB 7
SRR2086249 DRN CB 8
SRR2086250 DRN CB 9
SRR2086251 Liver BC 1
SRR2086252 Liver BC 2
SRR2086253 Liver BC 3
SRR2086254 Liver BC 4
SRR2086255 Liver BC 5
SRR2086256 Liver BC 6
SRR2086257 Liver BC 7
SRR2086258 Liver BC 8
SRR2086259 Liver CB 1
SRR2086260 Liver CB 2
SRR2086261 Liver CB 3
SRR2086262 Liver CB 4
SRR2086263 Liver CB 5
SRR2086264 Liver CB 6
SRR2086265 Liver CB 7
SRR2086266 Liver CB 8
SRR2086267 Muscle BC 1
SRR2086268 Muscle BC 2
SRR2086269 Muscle BC 3
SRR2086270 Muscle BC 4
SRR2086271 Muscle BC 5
SRR2086272 Muscle BC 6
SRR2086273 Muscle BC 7
SRR2086274 Muscle BC 8
SRR2086275 Muscle CB 1
SRR2086276 Muscle CB 2
SRR2086277 Muscle CB 3
SRR2086278 Muscle CB 4
SRR2086279 Muscle CB 5
SRR2086280 Muscle CB 6
SRR2086281 Muscle CB 7
SRR2086282 Muscle CB 8

Perez_2015

Accession Tissue Cross Replicate
SRR1952382 P8 Female CB 1
SRR1952383 P8 Female CB 2
SRR1952384 P8 Female CB 3
SRR1952385 P8 Female CB 4
SRR1952386 P8 Female CB 5
SRR1952387 P8 Female CB 6
SRR1952388 P8 Male CB 1
SRR1952389 P8 Male CB 2
SRR1952390 P8 Male CB 3
SRR1952391 P8 Male CB 4
SRR1952392 P8 Male CB 5
SRR1952393 P8 Male CB 6
SRR1952394 P8 Female BC 1
SRR1952395 P8 Female BC 2
SRR1952396 P8 Female BC 3
SRR1952397 P8 Female BC 4
SRR1952398 P8 Female BC 5
SRR1952399 P8 Female BC 6
SRR1952400 P8 Male BC 1
SRR1952401 P8 Male BC 2
SRR1952402 P8 Male BC 3
SRR1952403 P8 Male BC 4
SRR1952404 P8 Male BC 5
SRR1952405 P8 Male BC 6
SRR1952406 P60 Female CB 1
SRR1952407 P60 Female CB 2
SRR1952408 P60 Female CB 3
SRR1952409 P60 Female CB 4
SRR1952410 P60 Female CB 5
SRR1952411 P60 Female CB 6
SRR1952412 P60 Male CB 1
SRR1952413 P60 Male CB 2
SRR1952414 P60 Male CB 3
SRR1952415 P60 Male CB 4
SRR1952416 P60 Male CB 5
SRR1952417 P60 Male CB 6
SRR1952419 P60 Female BC 1
SRR1952420 P60 Female BC 2
SRR1952421 P60 Female BC 3
SRR1952422 P60 Female BC 4
SRR1952423 P60 Female BC 5
SRR1952424 P60 Female BC 6
SRR1952425 P60 Male BC 1
SRR1952426 P60 Male BC 2
SRR1952427 P60 Male BC 3
SRR1952428 P60 Male BC 4
SRR1952429 P60 Male BC 5
SRR1952430 P60 Male BC 6

Teichman_ESCs_NPCs

Accession Tissue Cross Replicate
SRR6330118 ESCs BC8
SRR6330119 ESCs CB9
SRR6330120 neural precursor cell (day 3) BC8
SRR6330121 neural precursor cell (day 3) CB9
SRR6330122 neural precursor cell (day 6) BC8
SRR6330123 neural precursor cell (day 6) CB9
SRR6330124 neural precursor cell (day 8) BC8
SRR6330125 neural precursor cell (day 8) CB9

Data Processing

1. Download FASTQ files from EMBL-EBI European Nucleotide Archive

Dataset WGET commands
Andergassen_2015 Andergassen_2015.wget.sh
Babak_2015 Babak_2015.wget.sh
Bonthuis_2015 Bonthuis_2015.wget.sh
Perez_2015 Perez_2015.wget.sh
Teichman_ESCs_NPCs Teichman_ESCs_NPCs.wget.sh

2. Trim low quality bases and adapter sequences

Using TrimGalore! run via the ClusterFlow pipeline tool. Single and paired-end reads are automatically determined and run accordingly.

cf trim_galore *.fq.gz or cf trim_galore *.fastq.gz

3. Reference Genome Preparation

i. Download the mouse SNPs file from the Mouse Genome Project at Sanger

[mgp.v5.merged.snps_all.dbSNP142.vcf.gz]

ii. Genome Prep

Assumes a directory, GRCm38_fasta, containing the BL6 GRCm38 reference genome fasta files

BL6 Vs CAST

SNPsplit_genome_preparation --vcf_file mgp.v5.merged.snps_all.dbSNP142.vcf.gz --reference_genome GRCm38_fasta/ --strain CAST_EiJ

CAST Vs FVB (based on GRCm38)

SNPsplit_genome_preparation --vcf_file mgp.v5.merged.snps_all.dbSNP142.vcf.gz --reference_genome GRCm38_fasta/ --strain CAST_EiJ --strain2 FVB_NJ --dual_hybrid

4. Align with HISAT2

a. BL6 vs CAST crosses

cf --genome CAST_EiJ_N-masked hisat2 *.fq.gz

b. BL6 vs CAST crosses

cf --genome CAST_EiJ_FVB_NJ_dual_hybrid.based_on_GRCm38_N-masked hisat2 *.fq.gz

5. Sort alignment files by name

cf --params byname samtools_sort *N-masked_hisat2.bam

6. SNPSplit

Run via a custom clusterflow module [SNPSplit.cfmod.pl]](Code/SNPSplit.cfmod.pl), or command line.

a. Via clusterflow

For single-end reads: BL6 Vs CAST

cf --genome all_SNPs_CAST_EiJ_GRCm38 --params sorted,paired SNPSplit *N-masked_hisat2_srtd.bam

For single-end reads: CAST Vs FVB

cf --genome all_FVB_NJ_SNPs_CAST_EiJ_reference.based_on_GRCm38 --params sorted SNPSplit *N-masked_hisat2_srtd.bam

For Paired-end reads: BL6 Vs CAST

cf --genome all_SNPs_CAST_EiJ_GRCm38 --params sorted,paired SNPSplit *N-masked_hisat2_srtd.bam

For Paired-end reads: CAST Vs FVB

cf --genome all_FVB_NJ_SNPs_CAST_EiJ_reference.based_on_GRCm38 --params sorted,paired SNPSplit *N-masked_hisat2_srtd.bam

b. Via command line

For BL6 Vs CAST crosses

SNPFILE="all_SNPs_CAST_EiJ_GRCm38.txt.gz"

For CAST Vs FVB crosses

SNPFILE="all_FVB_NJ_SNPs_CAST_EiJ_reference.based_on_GRCm38.txt.gz"

Run for all alignment files in a directory

for i in *N-masked_hisat2_srtd.bam;
  do

    SNPsplit --paired --no_sort --snp_file ${SNPFILE} ${i} &> ${i/.bam/.snpsplit.log}

  done

7. FeatureCounts

Gene counts from alignment files are calculated using featureCounts

cf --genome GRCm38 featureCounts *genome[12].bam

8. Count Matrix Generation

A custom Rscript DESeq2_featureCounts_2_CountsTables.R is used to make a single counts table from the individual featureCount files.

Replace FOLDERNAME with the directory name containing the featureCount files.

Rscript DESeq2_featureCounts_2_CountsTables.R FOLDERNAME

Allelic Bias Analysis

R code available in [ExploreBiasVsDist.R]. This is exploratory code and needs tidying up before publishing. Code works, but not commented or structured in a sensible way.

Isolde Processed Data

Tissue ASE BA UN
ARN [ISoLDE_result_ASE_04-07-2020_18-27-13.tsv] [ISoLDE_result_BA_04-07-2020_18-27-13.tsv] [ISoLDE_result_UN_04-07-2020_18-27-13.tsv]
DRN [ISoLDE_result_ASE_04-20-2020_16-15-29.tsv] [ISoLDE_result_BA_04-20-2020_16-15-29.tsv] [ISoLDE_result_UN_04-20-2020_16-15-29.tsv]
Liver [ISoLDE_result_ASE_04-08-2020_11-40-18.tsv]  [ISoLDE_result_BA_04-08-2020_11-40-18.tsv] [ISoLDE_result_UN_04-08-2020_11-40-18.tsv]
Muscle [ISoLDE_result_ASE_04-08-2020_15-28-14.tsv]  [ISoLDE_result_BA_04-08-2020_15-28-14.tsv]  [ISoLDE_result_UN_04-08-2020_15-28-14.tsv]
P60_Cerebellum [ISoLDE_result_ASE_06-23-2020_16-27-10.tsv]  [ISoLDE_result_BA_06-23-2020_16-27-10.tsv]  [ISoLDE_result_UN_06-23-2020_16-27-10.tsv]
P8_Cerebellum [ISoLDE_result_ASE_06-23-2020_16-21-15.tsv]  [ISoLDE_result_BA_06-23-2020_16-21-15.tsv] [ISoLDE_result_UN_06-23-2020_16-21-15.tsv]

Bias Vs Distance for ICR

[PDF Version]

Genome-wide Bias Vs Distance

Needs completing

Software Versions

Software Version Citation
ClusterFlow v0.5 dev [DOI]
TrimGalore! v0.4.1
HISAT2 v2.1.0 [DOI]
samtools v1.9 [DOI]
featureCounts (subread) v1.5.0-p2 [DOI]
SNPsplit v0.3.4 [DOI

Links

Description URL
Preprint https://doi.org/10.1101/2022.08.21.504536
Publication https://doi.org/10.7554/eLife.83364

Contact

Contact Russell S. Hamilton (darogan@gmail.com)