GRIAC/system_genetics
7
2
Fork 3
Code Issues Pull Requests 1 Releases Wiki Activity

Small snippet that generates a genome index and aligns the RNAseq reads. #2

Merged
J.v.N. merged 2 commits from step3/star-alignment-snippet into master 2021-02-15 17:09:32 +01:00
Conversation 3 Commits 2 Files Changed 1 +53
H.C. Donker commented 2021-02-09 17:36:21 +01:00
Owner
Copy Link

Minimal example of alignment using STAR:

  • Generates genome index.
  • Performs the alignment.
Minimal example of alignment using `STAR`: - Generates genome index. - Performs the alignment.
H.C. Donker added 1 commit 2021-02-09 17:36:22 +01:00
Small snippet that generates a genome index and aligns the RNAseq reads. ef4a25f54b
H.C. Donker added 1 commit 2021-02-11 10:47:32 +01:00
Tailored storage locations to calculon. d71460a7dd
H.C. Donker requested review from A.K. Saikumar Jayalatha 2021-02-12 11:59:24 +01:00
H.C. Donker requested review from J.v.N. 2021-02-12 11:59:40 +01:00
J.v.N. commented 2021-02-12 14:19:27 +01:00
Owner
Copy Link

Looks quite well.

Looks quite well.
H.C. Donker force-pushed step3/star-alignment-snippet from d71460a7dd to 1037d154e3 2021-02-12 14:46:31 +01:00 Compare
A.K. Saikumar Jayalatha approved these changes 2021-02-12 15:29:05 +01:00
rnaseq/step3_align/snippet.sh Outdated
@@ -0,0 +21,4 @@
# Storage location reference data (in this case on calculon).
REFERENCE_DATA="/groups/umcg-griac/prm02/rawdata/reference/genome"
GTF_FILE="${REFERENCE_DATA}/Homo_sapiens.GRCh38.100.gtf.gz"
A.K. Saikumar Jayalatha commented 2021-02-12 15:23:15 +01:00
Owner
Copy Link
Alternative reference file use : gencode.v19.annotation.gtf/gff3
##### Alternative reference file use : gencode.v19.annotation.gtf/gff3
J.v.N. commented 2021-02-12 16:42:42 +01:00
Owner
Copy Link

Should I upload these to the server as well? Also, what is the reason to use another reference file than the ones we already have?

Should I upload these to the server as well? Also, what is the reason to use another reference file than the ones we already have?
rnaseq/step3_align/snippet.sh Outdated
@@ -0,0 +22,4 @@
# Storage location reference data (in this case on calculon).
REFERENCE_DATA="/groups/umcg-griac/prm02/rawdata/reference/genome"
GTF_FILE="${REFERENCE_DATA}/Homo_sapiens.GRCh38.100.gtf.gz"
FASTA_FILE="${REFERENCE_DATA}/Homo_sapiens.GRCh38.dna.primary_assembly.fa.gz"
A.K. Saikumar Jayalatha commented 2021-02-12 15:24:33 +01:00
Owner
Copy Link
Alternative assembly file used : Homo_sapiens_assembly19.fasta
##### Alternative assembly file used : Homo_sapiens_assembly19.fasta
rnaseq/step3_align/snippet.sh Outdated
@@ -0,0 +28,4 @@
--runThreadN 8 \
--runMode genomeGenerate \
--readFilesCommand zcat \
--sjdbOverhang 100 \
A.K. Saikumar Jayalatha commented 2021-02-12 14:07:10 +01:00
Owner
Copy Link
sjdbOverhang 150
This was chosen based on the base pair reads for my analysis
##### sjdbOverhang 150 ##### This was chosen based on the base pair reads for my analysis
A.K. Saikumar Jayalatha commented 2021-02-12 15:27:15 +01:00
Owner
Copy Link

Alternative cut-off used : 150

This is also due to how my samples were sequenced (low-input method)

#### Alternative cut-off used : 150 #### This is also due to how my samples were sequenced (low-input method)
A.K. Saikumar Jayalatha commented 2021-02-12 15:27:48 +01:00
Owner
Copy Link

Alternative sjbOverhang cut-off used : 150

Alternative sjbOverhang cut-off used : 150
rnaseq/step3_align/snippet.sh Outdated
@@ -0,0 +32,4 @@
--genomeFastaFiles ${FASTA_FILE} \
--sjdbGTFfile ${GTF_FILE} \
--genomeDir ${GENOME_INDEX}
A.K. Saikumar Jayalatha commented 2021-02-12 15:28:43 +01:00
Owner
Copy Link
Alternative sjdbOverhang used : 150
This is also chosen due to the low-input RNA for sequencing.
##### Alternative sjdbOverhang used : 150 ##### This is also chosen due to the low-input RNA for sequencing.
J.v.N. approved these changes 2021-02-12 16:43:12 +01:00
rnaseq/step3_align/snippet.sh Outdated
@@ -0,0 +2,4 @@
#
# Align reads against reference genome.
STORAGE="/groups/umcg-griac/tmp04/rawdata/$(whoami)/step3"
J.v.N. commented 2021-02-12 14:16:23 +01:00
Owner
Copy Link

I think /groups/umcg-griac/tmp04/rawdata/$(whoami) should be a separate variable named 'project_directory' (or similar). This implies that the project has to be here.

I think `/groups/umcg-griac/tmp04/rawdata/$(whoami)` should be a separate variable named 'project_directory' (or similar). This implies that the project has to be here.
rnaseq/step3_align/snippet.sh Outdated
@@ -0,0 +8,4 @@
mkdir -p "${GENOME_INDEX}"
# Store the generated `Aligned.sortedByCoord.out.bam` in this dir.
ALIGNMENT_OUTPUT="${STORAGE}/alignment"
mkdir -p "${OUTPUT}"
J.v.N. commented 2021-02-12 14:13:53 +01:00
Owner
Copy Link

${OUTPUT} is unknown - should probably be ${ALIGNMENT_OUTPUT}

${OUTPUT} is unknown - should probably be ${ALIGNMENT_OUTPUT}
rnaseq/step3_align/snippet.sh Outdated
@@ -0,0 +21,4 @@
# Storage location reference data (in this case on calculon).
REFERENCE_DATA="/groups/umcg-griac/prm02/rawdata/reference/genome"
GTF_FILE="${REFERENCE_DATA}/Homo_sapiens.GRCh38.100.gtf.gz"
J.v.N. commented 2021-02-12 14:18:27 +01:00
Owner
Copy Link

I am unsure if STAR will decompress these files. Should I decompress them? Also, make sure the user notes the genome version.

I am unsure if STAR will decompress these files. Should I decompress them? Also, make sure the user notes the genome version.
J.v.N. added 1 commit 2021-02-15 17:08:37 +01:00
Merge branch 'master' into step3/star-alignment-snippet 65d7193848
J.v.N. merged commit 59f1b425b1 into master 2021-02-15 17:09:32 +01:00
Sign in to join this conversation.
Reviewers
No Reviewers
A.K. Saikumar Jayalatha
J.v.N.
Labels
No items
No Label
Milestone
No items
No Milestone
Assignees
Clear assignees
P280381 A.K. Saikumar Jayalatha P282473 C. Qi P271316 H.C. Donker P300299 J.v.N. P278949 M. Berg
No Assignees
3 Participants
Notifications
Due Date
No due date set.
Dependencies

No dependencies set.

Reference: GRIAC/system_genetics#2
No description provided.
Delete Branch "step3/star-alignment-snippet"

Deleting a branch is permanent. Although the deleted branch may continue to exist for a short time before it actually gets removed, it CANNOT be undone in most cases. Continue?