doubts about assembling paired and unpaired reads

[Arely Lechuga Jiménez]

Hi!!

I hope you finds you well.

i'm new Trinity user, so I tiried to assembled my reads both together paired and unpaired using the indications at the manual but Trinity makrs an error. " you cannot assemble unpaired reads using --left and --rigth"

So, What is the correct way to run trinity to assemble paired and unpaired readings together?

I obted for assembling first paired reads and later unpaired reads but I cannot run Trinity with unpaired reads because it has an error:

Error, found read_type 2 but expecting read_type 1
Error, cmd: seqtk-trinity seq -A -R 1  <(gunzip -c /mnt/9C908FFC908FDB62/Arely/Metatranscriptoma_BP8/Sortmerna_Filter/2_BP8_MT/unpaired_DR18_R2_non-ribosomal_2.fastq.gz) >> single.fa died with ret 512 at /home/proyectoh/Programas/trinityrnaseq-v2.12.0/util/insilico_read_normalization.pl line 793.
Error, cmd: /home/proyectoh/Programas/trinityrnaseq-v2.12.0/util/insilico_read_normalization.pl --seqType fq --JM 126G  --max_cov 200 --min_cov 1 --CPU 16 --output /home/arely/MT_BP8/Ensamble/unpaired_trinity/insilico_read_normalization --max_CV 10000  --single /mnt/9C908FFC908FDB62/Arely/Metatranscriptoma_BP8/Sortmerna_Filter/2_BP8_MT/unpaired_DR10_R1_non-ribosomal_1.fastq.gz,/mnt/9C908FFC908FDB62/Arely/Metatranscriptoma_BP8/Sortmerna_Filter/2_BP8_MT/unpaired_DR10_R1_non-ribosomal_2.fastq.gz,/mnt/9C908FFC908FDB62/Arely/Metatranscriptoma_BP8/Sortmerna_Filter/2_BP8_MT/unpaired_DR13_R1_non-ribosomal_1.fastq.gz,/mnt/9C908FFC908FDB62/Arely/Metatranscriptoma_BP8/Sortmerna_Filter/2_BP8_MT/unpaired_DR13_R1_non-ribosomal_2.fastq.gz,/mnt/9C908FFC908FDB62/Arely/Metatranscriptoma_BP8/Sortmerna_Filter/2_BP8_MT/unpaired_DR14_R1_non-ribosomal_1.fastq.gz,/mnt/9C908FFC908FDB62/Arely/Metatranscriptoma_BP8/Sortmerna_Filter/2_BP8_MT/unpaired_DR14_R1_non-ribosomal_2.fastq.gz,/mnt/9C908FFC908FDB62/Arely/Metatranscriptoma_BP8/Sortmerna_Filter/2_BP8_MT/unpaired_DR02_R1_non-ribosomal_1.fastq.gz,/mnt/9C908FFC908FDB62/Arely/Metatranscriptoma_BP8/Sortmerna_Filter/2_BP8_MT/unpaired_DR02_R1_non-ribosomal_2.fastq.gz,/mnt/9C908FFC908FDB62/Arely/Metatranscriptoma_BP8/Sortmerna_Filter/2_BP8_MT/unpaired_DR03_R1_non-ribosomal_1.fastq.gz,/mnt/9C908FFC908FDB62/Arely/Metatranscriptoma_BP8/Sortmerna_Filter/2_BP8_MT/unpaired_DR03_R1_non-ribosomal_2.fastq.gz,/mnt/9C908FFC908FDB62/Arely/Metatranscriptoma_BP8/Sortmerna_Filter/2_BP8_MT/unpaired_DR06_R1_non-ribosomal_1.fastq.gz,/mnt/9C908FFC908FDB62/Arely/Metatranscriptoma_BP8/Sortmerna_Filter/2_BP8_MT/unpaired_DR06_R1_non-ribosomal_2.fastq.gz,/mnt/9C908FFC908FDB62/Arely/Metatranscriptoma_BP8/Sortmerna_Filter/2_BP8_MT/unpaired_DR04_R1_non-ribosomal_1.fastq.gz,/mnt/9C908FFC908FDB62/Arely/Metatranscriptoma_BP8/Sortmerna_Filter/2_BP8_MT/unpaired_DR04_R1_non-ribosomal_2.fastq.gz,/mnt/9C908FFC908FDB62/Arely/Metatranscriptoma_BP8/Sortmerna_Filter/2_BP8_MT/unpaired_DR05_R1_non-ribosomal_1.fastq.gz,/mnt/9C908FFC908FDB62/Arely/Metatranscriptoma_BP8/Sortmerna_Filter/2_BP8_MT/unpaired_DR05_R1_non-ribosomal_2.fastq.gz,/mnt/9C908FFC908FDB62/Arely/Metatranscriptoma_BP8/Sortmerna_Filter/2_BP8_MT/unpaired_DR18_R1_non-ribosomal_1.fastq.gz,/mnt/9C908FFC908FDB62/Arely/Metatranscriptoma_BP8/Sortmerna_Filter/2_BP8_MT/unpaired_DR18_R1_non-ribosomal_2.fastq.gz,/mnt/9C908FFC908FDB62/Arely/Metatranscriptoma_BP8/Sortmerna_Filter/2_BP8_MT/unpaired_DR18_R2_non-ribosomal_2.fastq.gz,/mnt/9C908FFC908FDB62/Arely/Metatranscriptoma_BP8/Sortmerna_Filter/2_BP8_MT/unpaired_DR10_R2_non-ribosomal_1.fastq.gz,/mnt/9C908FFC908FDB62/Arely/Metatranscriptoma_BP8/Sortmerna_Filter/2_BP8_MT/unpaired_DR10_R2_non-ribosomal_2.fastq.gz,/mnt/9C908FFC908FDB62/Arely/Metatranscriptoma_BP8/Sortmerna_Filter/2_BP8_MT/unpaired_DR13_R2_non-ribosomal_1.fastq.gz,/mnt/9C908FFC908FDB62/Arely/Metatranscriptoma_BP8/Sortmerna_Filter/2_BP8_MT/unpaired_DR13_R2_non-ribosomal_2.fastq.gz,/mnt/9C908FFC908FDB62/Arely/Metatranscriptoma_BP8/Sortmerna_Filter/2_BP8_MT/unpaired_DR14_R2_non-ribosomal_1.fastq.gz,/mnt/9C908FFC908FDB62/Arely/Metatranscriptoma_BP8/Sortmerna_Filter/2_BP8_MT/unpaired_DR14_R2_non-ribosomal_2.fastq.gz,/mnt/9C908FFC908FDB62/Arely/Metatranscriptoma_BP8/Sortmerna_Filter/2_BP8_MT/unpaired_DR02_R2_non-ribosomal_1.fastq.gz,/mnt/9C908FFC908FDB62/Arely/Metatranscriptoma_BP8/Sortmerna_Filter/2_BP8_MT/unpaired_DR02_R2_non-ribosomal_2.fastq.gz,/mnt/9C908FFC908FDB62/Arely/Metatranscriptoma_BP8/Sortmerna_Filter/2_BP8_MT/unpaired_DR03_R2_non-ribosomal_1.fastq.gz,/mnt/9C908FFC908FDB62/Arely/Metatranscriptoma_BP8/Sortmerna_Filter/2_BP8_MT/unpaired_DR03_R2_non-ribosomal_2.fastq.gz,/mnt/9C908FFC908FDB62/Arely/Metatranscriptoma_BP8/Sortmerna_Filter/2_BP8_MT/unpaired_DR06_R2_non-ribosomal_1.fastq.gz,/mnt/9C908FFC908FDB62/Arely/Metatranscriptoma_BP8/Sortmerna_Filter/2_BP8_MT/unpaired_DR06_R2_non-ribosomal_2.fastq.gz,/mnt/9C908FFC908FDB62/Arely/Metatranscriptoma_BP8/Sortmerna_Filter/2_BP8_MT/unpaired_DR04_R2_non-ribosomal_1.fastq.gz,/mnt/9C908FFC908FDB62/Arely/Metatranscriptoma_BP8/Sortmerna_Filter/2_BP8_MT/unpaired_DR04_R2_non-ribosomal_2.fastq.gz,/mnt/9C908FFC908FDB62/Arely/Metatranscriptoma_BP8/Sortmerna_Filter/2_BP8_MT/unpaired_DR05_R2_non-ribosomal_1.fastq.gz,/mnt/9C908FFC908FDB62/Arely/Metatranscriptoma_BP8/Sortmerna_Filter/2_BP8_MT/unpaired_DR05_R2_non-ribosomal_1.fastq.gz,/mnt/9C908FFC908FDB62/Arely/Metatranscriptoma_BP8/Sortmerna_Filter/2_BP8_MT/unpaired_DR18_R2_non-ribosomal_1.fastq.gz died with ret 512 at /home/proyectoh/Programas/trinityrnaseq-v2.12.0/Trinity line 2869.
    main::process_cmd("/home/proyectoh/Programas/trinityrnaseq-v2.12.0/util/insilico"...) called at /home/proyectoh/Programas/trinityrnaseq-v2.12.0/Trinity line 3422
    main::normalize("/home/arely/MT_BP8/Ensamble/unpaired_trinity/insilico_read_no"..., 200, ARRAY(0x55a41d38e420)) called at /home/proyectoh/Programas/trinityrnaseq-v2.12.0/Trinity line 3362
    main::run_normalization(200, ARRAY(0x55a41d38e420)) called at /home/proyectoh/Programas/trinityrnaseq-v2.12.0/Trinity line 1389

I changed the order of my read from the --samples_file (first forward and under reverse) but it shows a similar error

-------------------------------------------
----------- Jellyfish  --------------------
-- (building a k-mer catalog from reads) --
-------------------------------------------

CMD: jellyfish count -t 16 -m 25 -s 100000000  --canonical  single.fa
^CError, cmd: jellyfish count -t 16 -m 25 -s 100000000  --canonical  single.fa died with ret 2 at /home/proyectoh/Programas/trinityrnaseq-v2.12.0/util/insilico_read_normalization.pl line 793.
Error, cmd: /home/proyectoh/Programas/trinityrnaseq-v2.12.0/util/insilico_read_normalization.pl --seqType fq --JM 126G  --max_cov 200 --min_cov 1 --CPU 16 --output /home/arely/MT_BP8/Ensamble/unpaired_R1_trinity/insilico_read_normalization --max_CV 10000  --single /mnt/9C908FFC908FDB62/Arely/Metatranscriptoma_BP8/Sortmerna_Filter/2_BP8_MT/unpaired_DR10_R1_non-ribosomal_1.fastq.gz,/mnt/9C908FFC908FDB62/Arely/Metatranscriptoma_BP8/Sortmerna_Filter/2_BP8_MT/unpaired_DR10_R1_non-ribosomal_2.fastq.gz,/mnt/9C908FFC908FDB62/Arely/Metatranscriptoma_BP8/Sortmerna_Filter/2_BP8_MT/unpaired_DR13_R1_non-ribosomal_1.fastq.gz,/mnt/9C908FFC908FDB62/Arely/Metatranscriptoma_BP8/Sortmerna_Filter/2_BP8_MT/unpaired_DR13_R1_non-ribosomal_2.fastq.gz,/mnt/9C908FFC908FDB62/Arely/Metatranscriptoma_BP8/Sortmerna_Filter/2_BP8_MT/unpaired_DR14_R1_non-ribosomal_1.fastq.gz,/mnt/9C908FFC908FDB62/Arely/Metatranscriptoma_BP8/Sortmerna_Filter/2_BP8_MT/unpaired_DR14_R1_non-ribosomal_2.fastq.gz,/mnt/9C908FFC908FDB62/Arely/Metatranscriptoma_BP8/Sortmerna_Filter/2_BP8_MT/unpaired_DR02_R1_non-ribosomal_1.fastq.gz,/mnt/9C908FFC908FDB62/Arely/Metatranscriptoma_BP8/Sortmerna_Filter/2_BP8_MT/unpaired_DR02_R1_non-ribosomal_2.fastq.gz,/mnt/9C908FFC908FDB62/Arely/Metatranscriptoma_BP8/Sortmerna_Filter/2_BP8_MT/unpaired_DR03_R1_non-ribosomal_1.fastq.gz,/mnt/9C908FFC908FDB62/Arely/Metatranscriptoma_BP8/Sortmerna_Filter/2_BP8_MT/unpaired_DR03_R1_non-ribosomal_2.fastq.gz,/mnt/9C908FFC908FDB62/Arely/Metatranscriptoma_BP8/Sortmerna_Filter/2_BP8_MT/unpaired_DR06_R1_non-ribosomal_1.fastq.gz,/mnt/9C908FFC908FDB62/Arely/Metatranscriptoma_BP8/Sortmerna_Filter/2_BP8_MT/unpaired_DR06_R1_non-ribosomal_2.fastq.gz,/mnt/9C908FFC908FDB62/Arely/Metatranscriptoma_BP8/Sortmerna_Filter/2_BP8_MT/unpaired_DR04_R1_non-ribosomal_1.fastq.gz,/mnt/9C908FFC908FDB62/Arely/Metatranscriptoma_BP8/Sortmerna_Filter/2_BP8_MT/unpaired_DR04_R1_non-ribosomal_2.fastq.gz,/mnt/9C908FFC908FDB62/Arely/Metatranscriptoma_BP8/Sortmerna_Filter/2_BP8_MT/unpaired_DR05_R1_non-ribosomal_1.fastq.gz,/mnt/9C908FFC908FDB62/Arely/Metatranscriptoma_BP8/Sortmerna_Filter/2_BP8_MT/unpaired_DR05_R1_non-ribosomal_2.fastq.gz,/mnt/9C908FFC908FDB62/Arely/Metatranscriptoma_BP8/Sortmerna_Filter/2_BP8_MT/unpaired_DR18_R1_non-ribosomal_1.fastq.gz,/mnt/9C908FFC908FDB62/Arely/Metatranscriptoma_BP8/Sortmerna_Filter/2_BP8_MT/unpaired_DR18_R1_non-ribosomal_2.fastq.gz died with ret 512 at /home/proyectoh/Programas/trinityrnaseq-v2.12.0/Trinity line 2869.
    main::process_cmd("/home/proyectoh/Programas/trinityrnaseq-v2.12.0/util/insilico"...) called at /home/proyectoh/Programas/trinityrnaseq-v2.12.0/Trinity line 3422
    main::normalize("/home/arely/MT_BP8/Ensamble/unpaired_R1_trinity/insilico_read"..., 200, ARRAY(0x563e1a3a80a0)) called at /home/proyectoh/Programas/trinityrnaseq-v2.12.0/Trinity line 3362
    main::run_normalization(200, ARRAY(0x563e1a3a80a0)) called at /home/proyectoh/Programas/trinityrnaseq-v2.12.0/Trinity line 1389


So, What is the properly way to assemble both paired and unpaired reads (comming from PE data) together? and how a I assemble unpaired forward and reverse reads ?

Thanks,

[Brian Haas]

Here's our faq on mixing PE / SE data:

https://github.com/trinityrnaseq/trinityrnaseq/wiki/How-do-I-combine-reads%3F

[Arely Lechuga Jiménez]

Thanks