Reference and Resources

Nature Protocols volume 8, pages 1494-1512 (2013)

AWS instance: m4.xlarge

Directory contents

tuxedo2_HSB_Spombe$ ls -l

-rw-rw-r– 1 ubuntu ubuntu 7830629 Mar 5 02:01 S_pombe_refTrans.fasta

-rw-rw-r– 1 ubuntu ubuntu 175846179 Mar 5 02:00 Sp.ds.1M.left.fq

-rw-rw-r– 1 ubuntu ubuntu 175846179 Mar 5 02:00 Sp.ds.1M.right.fq

-rw-rw-r– 1 ubuntu ubuntu 175736042 Mar 5 02:00 Sp.hs.1M.left.fq

-rw-rw-r– 1 ubuntu ubuntu 175736042 Mar 5 02:00 Sp.hs.1M.right.fq

-rw-rw-r– 1 ubuntu ubuntu 175741215 Mar 5 02:00 Sp.log.1M.left.fq

-rw-rw-r– 1 ubuntu ubuntu 175741215 Mar 5 02:00 Sp.log.1M.right.fq

-rw-rw-r– 1 ubuntu ubuntu 175899533 Mar 5 02:00 Sp.plat.1M.left.fq

-rw-rw-r– 1 ubuntu ubuntu 175899533 Mar 5 02:00 Sp.plat.1M.right.fq

drwxrwxr-x 2 ubuntu ubuntu 4096 Mar 5 02:44 SpHisat_index

drwxrwxr-x 2 ubuntu ubuntu 4096 Mar 5 02:56 Aligned_Stats

drwxrwxr-x 2 ubuntu ubuntu 4096 Mar 5 03:03 mAligned_bam

drwxrwxr-x 2 ubuntu ubuntu 4096 Mar 5 02:54 Aligned_sam

Assemble the transcriptome using StringTie

tuxedo2_HSB_Spombe$

stringtie Aligned_bam/Sp_ds.sorted.bam -p 8 –rf -o Assembled_transcripts/Sp_ds.gtf

stringtie Aligned_bam/Sp_hs.sorted.bam -p 8 –rf -o Assembled_transcripts/Sp_hs.gtf

stringtie Aligned_bam/Sp_plat.sorted.bam -p 8 –rf -o Assembled_transcripts/Sp_plat.gtf

stringtie Aligned_bam/Sp_log.sorted.bam -p 8 –rf -o Assembled_transcripts/Sp_log.gtf

Merge the assembled transcripts into a single GTF file

ls -l Assembled_transcripts/

-rw-rw-r– 1 ubuntu ubuntu 1219771 Mar 5 03:43 Sp_ds.gtf

-rw-rw-r– 1 ubuntu ubuntu 1081512 Mar 5 03:44 Sp_hs.gtf

-rw-rw-r– 1 ubuntu ubuntu 1232203 Mar 5 03:45 Sp_log.gtf

-rw-rw-r– 1 ubuntu ubuntu 1223911 Mar 5 03:44 Sp_plat.gtf

ls Assembled_transcripts/*.gtf > mergelist.txt

cat mergelist.txt

Assembled_transcripts/Sp_ds.gtf

Assembled_transcripts/Sp_hs.gtf

Assembled_transcripts/Sp_log.gtf

Assembled_transcripts/Sp_plat.gtf

stringtie –merge -p 8 -o stringtie_merged.gtf mergelist.txt

Prepare the merged GTF files for differential analysis using Ballgown

stringtie -e -B -p 8 -G stringtie_merged.gtf -o ballgown/Sp_ds/Sp_ds.gtf Aligned_bam/Sp_ds.sorted.bam

stringtie -e -B -p 8 -G stringtie_merged.gtf -o ballgown/Sp_hs/Sp_hs.gtf Aligned_bam/Sp_hs.sorted.bam

stringtie -e -B -p 8 -G stringtie_merged.gtf -o ballgown/Sp_plat/Sp_plat.gtf Aligned_bam/Sp_plat.sorted.bam

stringtie -e -B -p 8 -G stringtie_merged.gtf -o ballgown/Sp_log/Sp_log.gtf Aligned_bam/Sp_log.sorted.bam

ls -l ballgown/Sp_ds/

-rw-rw-r– 1 ubuntu ubuntu 1460476 Mar 5 05:15 Sp_ds.gtf

-rw-rw-r– 1 ubuntu ubuntu 49218 Mar 5 05:15 e2t.ctab

-rw-rw-r– 1 ubuntu ubuntu 364883 Mar 5 05:15 e_data.ctab

-rw-rw-r– 1 ubuntu ubuntu 664 Mar 5 05:15 i2t.ctab

-rw-rw-r– 1 ubuntu ubuntu 2516 Mar 5 05:15 i_data.ctab

-rw-rw-r– 1 ubuntu ubuntu 407989 Mar 5 05:15 t_data.ctab

ls -l ballgown/Sp_hs/

-rw-rw-r– 1 ubuntu ubuntu 1458154 Mar 5 05:16 Sp_hs.gtf

-rw-rw-r– 1 ubuntu ubuntu 49218 Mar 5 05:16 e2t.ctab

-rw-rw-r– 1 ubuntu ubuntu 362372 Mar 5 05:16 e_data.ctab

-rw-rw-r– 1 ubuntu ubuntu 664 Mar 5 05:16 i2t.ctab

-rw-rw-r– 1 ubuntu ubuntu 2483 Mar 5 05:16 i_data.ctab

-rw-rw-r– 1 ubuntu ubuntu 407244 Mar 5 05:16 t_data.ctab

ls -l ballgown/Sp_log/

-rw-rw-r– 1 ubuntu ubuntu 1458521 Mar 5 05:17 Sp_log.gtf

-rw-rw-r– 1 ubuntu ubuntu 49218 Mar 5 05:17 e2t.ctab

-rw-rw-r– 1 ubuntu ubuntu 364590 Mar 5 05:17 e_data.ctab

-rw-rw-r– 1 ubuntu ubuntu 664 Mar 5 05:17 i2t.ctab

-rw-rw-r– 1 ubuntu ubuntu 2492 Mar 5 05:17 i_data.ctab

-rw-rw-r– 1 ubuntu ubuntu 407658 Mar 5 05:17 t_data.ctab

ls -l ballgown/Sp_plat/

-rw-rw-r– 1 ubuntu ubuntu 1460785 Mar 5 05:16 Sp_plat.gtf

-rw-rw-r– 1 ubuntu ubuntu 49218 Mar 5 05:16 e2t.ctab

-rw-rw-r– 1 ubuntu ubuntu 364635 Mar 5 05:16 e_data.ctab

-rw-rw-r– 1 ubuntu ubuntu 664 Mar 5 05:16 i2t.ctab

-rw-rw-r– 1 ubuntu ubuntu 2500 Mar 5 05:16 i_data.ctab

-rw-rw-r– 1 ubuntu ubuntu 408141 Mar 5 05:16 t_data.ctab

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