TagSeq pipeline trouble - Generating read-counts-per-gene about tag-based_rnaseq HOT 8 CLOSED

On Sep 10, 2019, at 4:20 PM, LibraLizard66 ***@***.***> wrote: Hi all, I'm trying to assay differential gene expression from 22 RNA samples, two of them sequenced with RNAseq and 20 of them sequenced with TagSeq. I used the RNAseq sequences to generate a reference transcriptome de novo via Trinity to compare the TagSeq sequences to. I've been following the pipeline listed here: https://github.com/z0on/tag-based_RNAseq/blob/master/tagSeq_processing_README.txt <https://github.com/z0on/tag-based_RNAseq/blob/master/tagSeq_processing_README.txt> Everything went smoothly until the "# generating read-counts-per gene" section. Here's a rundown of what I did and where things appeared wrong (Skip to Step 4 if you want to go straight to that). STEP 1 I used the command: grep ">" Trinity.fasta | perl -pe 's/^>(\S+)|(\S+)(i\d+)\s.+/$1|$2$3\t$1$2/' >transcriptome_seq2iso.tab in order to create a two-column tab-delimited table to give correspondence between entries in the transcriptome fasta file and genes (our transcriptome fasta file was named "Trinity.fasta", not "trinity.fasta" as per the readme file). The file "transcriptome_seq2iso.tab" was created. STEP 2 The pipeline afterwards lists some steps to take if you don't have any assembler-derived genes (cd-hit-est). As we had plenty of assembler-derived-genes, I planned on skipping it, but isogroup_namer.pl (in the next step) seems to depend on having ch-hit-est. Thus, I ran cd-hit-est using the following code: cdhit-est -i Trinity.fasta -o transcriptome_clust.fasta -c 0.99 -G 0 -aL 0.3 -aS 0.3 The following two files were created 1) transcriptome_clust.fasta and 2) transcriptome_clust.fasta.clustr STEP 3 To try to add cluster designations to the fasta headers, I used the code: /tag-based_RNAseq/isogroup_namer.pl transcriptome_clust.fasta transcriptome_clust.fasta.clstr >transcriptome_seq2gene.tab Note: The "/tag-based_RNAseq" portion of the code was to tell Ubuntu where isogroup_namer.pl was. The following four files were created: 1) transcriptome_clust_iso.fasta 2) transcriptome_clust_iso.seq2iso.tab 3) transcriptome_seq2gene.tab and 4) transcriptome_seq2iso.tab However, when I examined these files with nano, all of the files were empty except for transcriptome_seq2iso.tab (the 4th one), which, as far as I could tell, looked like it should. STEP 4 (THIS IS WHERE THINGS HAVE APPARENTLY GONE WRONG) To try to count the hits per isogroup, I ran the following code: /home/genomics/tag-based_RNAseq/samcount_launch_bt2.pl '.sam' /Data/devin/JA19193/transcriptome_seq2iso.tab > sc Note: "/home/genomics/tag-based_RNAseq" pointed Ubuntu to samcount_launch_bt2.pl, and "/Data/devin?JA19193" pointed Ubuntu towards transcriptome_seq2.iso.tab. After doing this, I used nano to edit sc to add the full path to samcount before each sample so Ubuntu could find samcount. For example: samcount.pl 10_merged.trim.sam /home/genomics/Data/devin/JA19193/transcriptome_seq2iso.tab aligner=bowtie2 >10_merged.trim.sam.counts became ~/tag-based_RNAseq/samcount.pl 10_merged.trim.sam /home/genomics/Data/devin/JA19193/transcriptome_seq2iso.tab aligner=bowtie2 >10_merged.trim.sam.counts Note: "10_merged" is the name of a TagSeq sample. I then executed sc using the following code: bash sc samcount ran, but in every line of output while it ran, the message, "[gene name] has no isogroup designation” appeared. I tried creating the sc file using the file "transcriptome_seq2gene.tab" (the 3rd file in Step 3 above) and running samcount with this modified sc, but received the same result. Thus, it appears that samcount is treating the only meaningful product file from isogroup_namer as an empty file. Can anyone give me some help? I'm running Ubuntu version 16.04. Thanks, -Devin — You are receiving this because you are subscribed to this thread. Reply to this email directly, view it on GitHub <#1?email_source=notifications&email_token=ABZUHGDQGMRQDNWO4ECE65LQJAFRHA5CNFSM4IVMWOB2YY3PNVWWK3TUL52HS4DFUVEXG43VMWVGG33NNVSW45C7NFSM4HKR3WTQ>, or mute the thread <https://github.com/notifications/unsubscribe-auth/ABZUHGCRTPOSD66W6DS677LQJAFRHANCNFSM4IVMWOBQ>.

On Sep 15, 2019, at 5:23 PM, LibraLizard66 ***@***.***> wrote: That got rid of the 'no isogroup designation' error while running. However, when running sc, I got many notices saying either "disregarding reads mapping to multiple isogroups" or "cannot find alignment score in" (sic). Do you think this is ok? For reference, I've provided the first ten lines of allcounts.txt that resulted from assembling all of the counts into a single table using: /home/genomics/tag-based_RNAseq/expression_compiler.pl *.sam.counts > allcounts.txt 10_merged.trim.sam.counts 11C_merged.trim.sam.counts 12C_merged.trim.sam.counts 13_merged.trim.sam.counts 14_merged.trim.sam.counts 15C_merged.trim.sam.counts 15_merged.trim.sam.counts 17_merged.trim.sam.counts 19_merged.trim.sam.counts 27_merged.trim.sam.counts 29_merged.trim.sam.counts 2_merged.trim.sam.counts 37_merged.trim.sam.counts 3C_merged.trim.sam.counts 4C_merged.tr$ TRINITY_DN0_c0_g1 1 0 3 1 1 3 1 0 1 2 3 1 0 2 6 3 1 2 2 0 TRINITY_DN0_c1_g1 130 90 78 81 58 184 110 78 157 69 69 91 108 64 97 125 131 96 213 154 TRINITY_DN10000_c0_g1 0 1 5 0 1 0 0 2 0 4 3 1 0 0 6 2 0 2 0 0 TRINITY_DN10000_c1_g1 0 0 0 0 1 0 0 0 0 2 0 1 0 0 0 0 0 0 0 0 TRINITY_DN10000_c1_g2 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 TRINITY_DN10000_c1_g3 0 0 0 0 0 0 0 0 2 0 1 1 0 0 0 0 0 0 0 0 TRINITY_DN10001_c0_g1 1 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 TRINITY_DN10002_c0_g1 5 5 9 3 4 3 3 6 5 6 7 9 2 4 3 3 3 3 6 3 — You are receiving this because you commented. Reply to this email directly, view it on GitHub <#1?email_source=notifications&email_token=ABZUHGFNFACSWXYEQX42UCTQJ2YXBA5CNFSM4IVMWOB2YY3PNVWWK3TUL52HS4DFVREXG43VMVBW63LNMVXHJKTDN5WW2ZLOORPWSZGOD6X2IDI#issuecomment-531604493>, or mute the thread <https://github.com/notifications/unsubscribe-auth/ABZUHGG4DQWLFLLI6BXYSIDQJ2YXBANCNFSM4IVMWOBQ>.