Does anyone have experience run STAR on Pacbio RNA sequencing

[Fujun Qin]

Hi, Alex,

Recently, I am trying to use STAR to run some Pacbio RNA sequencing. But I got some errors. 

"-bash-4.1$ more slurm-907216.out 

Aug 02 01:18:23 ..... started STAR run

Aug 02 01:18:23 ..... loading genome

Aug 02 01:18:24 ..... started mapping

EXITING because of FATAL ERROR in reads input: quality string length is not equa

l to sequence length

@SRR1853186.3

CCAGATGCTTAATATTTCGGATCAGACTTGCACTTCACGATAAGGCACTCGGAGCGTTATATTTGCGAATCACAGTTCGC

ATAAGATGTGATGTACTGCTGCTATACTTGGCACATGTTATATGTTGTGATTTTGTTTTCAGTGATCGACTGTCACGTTT

TTTGGACCGATTGTTGTTATAGCGTCTAAGTGACATACTACGTAGCATTGCTTACAGGCTACACATTGTTGACAGTAATG

TGCTTATGGTGATGATTTGATGCACAGATGATTATCTAGCAGCTTATGTCACGTACTCTACGTACATCTGTAATCATTAG

TTTAACGTTATTATCTAATTGGTATAAGTCTATGTTTCTACTGCAACGTAGCGATATTATTTAGATCAGTATTATTACTA

GTAGCGTATGTATGATTCGTTGTATGTAATTCGATATGTATATTTAGTGTTGAGCACTGAGTTATCTGATATTTATACTG

CGTGTATTCGATATGTGTTGCTGGATTGAGTGAGGTTTGTTTTTGTGCGGCACTATTTCATCGTACATTTTTGTATTGTA

GCAGTTGGTTGGTTGATCTTGACCTACTGTGACGAACTGCTCGATATTATTCGATTTATGATGCTGCTGAGTGGTCAGTT

TTCTGTGTTG

SOLUTION: fix your fastq file

Aug 02 01:18:24 ...... FATAL ERROR, exiting"

Do you know what is the problem ?

My command is "

/nv/vol80/hlilab/software/STAR-master/bin/Linux_x86_64_static/STAR --genomeDir /

nv/vol80/hlilab/SLC_ELK_project/download/SRA/genome/ --readFilesIn SRR1853186.fa

stq --runThreadN 2  "

Thanks,

Fujun

[Alexander Dobin]

Hi Fujun,

please follow this guide from PacBio on how to use STAR on Iso-Seq reads:

https://github.com/PacificBiosciences/cDNA_primer/wiki/Bioinfx-study:-Optimizing-STAR-aligner-for-Iso-Seq-data

Note that this will only work for Iso-Seq and circular consensus reads, not on raw reads.

Cheers

Alex

[Fujun Qin]

Thank you, Alex.

Let me introduce a little bit of what I am going to do.   I have a know and validated fusion RNA junction sequence.  I am using this fusion junction sequence or putative full length sequence of this fusion RNA to align to the Pacbio sequence. My aim is try to get positive long reads to support the full length of fusion. 

The first step for me is I will generate a artificial genome using my fusion junction sequence.  Should I use STARlong generate the genome ?  The parameters for generating genome is still similar as STAR ?  I know the mapping step is different.

Thanks,

Fujun

[Fujun Qin]

Hi, Alex,

I tried the following mapping command, but get error. Where should I change ?  Thanks.

/nv/vol80/hlilab/software/STAR-2.5.2a/bin/Linux_x86_64_static/STARlong --runMode

 alignReads --genomeDir /nv/vol80/hlilab/SLC_ELK_project/download/SRA/Pacbio/Raw

-MCF7/genome/ --readFilesIn /nv/vol80/hlilab/SLC_ELK_project/download/SRA/Pacbio

/Raw-MCF7/SRR1867931.fastq --outSAMattributes NH HI NM MD --readNameSeparator sp

ace --outFilterMultimapScoreRange 1 --outFilterMismatchNmax 2000 --scoreGapNonca

n -20 --scoreGapGCAG -4 --scoreGapATAC -8 --scoreDelOpen -1 --scoreDelBase -1 --

scoreInsOpen -1 --scoreInsBase -1 --alignEndsType Local --seedSearchStartLmax 50

 --seedPerReadNmax 100000 --seedPerWindowNmax 1000 --alignTranscriptsPerReadNmax

 100000 --alignTranscriptsPerWindowNmax 10000

This is the error 

Aug 03 14:31:27 ..... started STAR run

Aug 03 14:31:27 ..... loading genome

Aug 03 14:31:27 ..... started mapping

EXITING because of FATAL ERROR in reads input: short read sequence line: 1

Read Name=@SRR1867931.1

Read Sequence====

DEF_readNameLengthMax=50000

DEF_readSeqLengthMax=500000

Aug 03 14:31:28 ...... FATAL ERROR, exiting

FUJUN

[Fujun Qin]

[Fujun Qin]

-bash-4.1$  tail -n2 SRR1867931.fastq | hexdump -c

0000000   +   S   R   R   1   8   6   7   9   3   1   .   1   6   3   4

0000010   8   2  \n  \n                                                

0000014

-bash-4.1$ 

Hi, Alex,

I did above.

Thanks,

Fujun

[Rory Kirchner]

Hi Fujun,

Your FASTQ file is truncated— I bet when you downloaded it the download didn’t finish properly.

Best,

Rory

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[Hesham Gibriel]

Hi all, I actually had the same issue when running STAR with a PacBio RNAseq read. 

It seems that STAR does not accept reads longer than 650 bp.. When I first ran STAR on my input, I got the same error that you got, so I manually truncated the input length to 650 bp and STAR reads the input just fine.. So maybe STAR is not adjusted to long reads?

On Thursday, August 4, 2016 at 4:47:53 AM UTC+1, Rory Kirchner wrote:

Hi Fujun,

Your FASTQ file is truncated— I bet when you downloaded it the download didn’t finish properly.

Best,

Rory

On Aug 3, 2016, at 11:42 PM, Fujun Qin <qinf...@gmail.com> wrote:

-bash-4.1$  tail -n2 SRR1867931.fastq | hexdump -c

0000000   +   S   R   R   1   8   6   7   9   3   1   .   1   6   3   4

0000010   8   2  \n  \n                                                

0000014

-bash-4.1$ 

Hi, Alex,

I did above.

Thanks,

Fujun

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[Alexander Dobin]

Hi Hesham,

you would need to use STARlong for long reads, and tweak the parameters according to this guide:

https://github.com/Magdoll/cDNA_Cupcake/wiki/Best-practice-for-aligning-Iso-Seq-to-reference-genome:-minimap2,-deSALT,-GMAP,-STAR,-BLAT#refstar

Cheers

Alex