[Cufflinks] Multi-Hit error

[burt]

Hi, I'm running the latest build of Cufflinks (v1.0.3) on an Illumina paired-end RNA-seq library and its throwing me an error.

[chiakhb@pegasus biasCorrect_multiRead]$ cufflinks --output-dir brain_l8 --num-threads 10 --GTF-guide ~/ref_genome/hg18/misc/hg18.gtf --frag-bias-correct ~/ref_genome/hg18/misc/hg18.fa --multi-read-correct /mnt/ScratchPool/burt/MAQC_HBR/Tophat/brain_l8/accepted_hits.bam
cufflinks: /lib64/libz.so.1: no version information available (required by cufflinks)
You are using Cufflinks v1.0.3, which is the most recent release.
[17:57:40] Loading reference annotation.
[17:57:44] Inspecting reads and determining fragment length distribution.
> Processed 135078 loci. [*************************] 100%
> Map Properties:
> Total Map Mass: 7131868.26
> Number of Multi-Reads: 1 (with 458350 total hits)
> Read Type: 54bp x 54bp
> Fragment Length Distribution: Empirical (learned)
> Estimated Mean: 207.08
> Estimated Std Dev: 23.29
[18:00:08] Assembling transcripts and initializing abundances for multi-read correction.
> Processing Locus chr1:227643666-227710711 [** ] 8%
ERROR: Multi-Hit not found (227634990,227635040).

Anyone has encountered a similar error?

Regards
-burt

[shrutiisarda]

I got the same error, using the GFF guide option..

Here's what the error looks like..



chr9:130812841-130820923 Finding a maximum matching to collapse scaffolds
chr9:130812841-130820923 Will collapse 5 scaffolds
chr9:130812841-130820923 Starting new collapse round
chr9:130812841-130820923 Calculating scaffold densities
intron: [130820321-130820447], 0
intron: [130820321-130820447], 0
intron: [130820321-130820447], 0
intron: [130820321-130820447], 0
intron: [130820321-130820447], 0
intron: [130820321-130820447], 0
intron: [130820321-130820447], 0
intron: [130820321-130820447], 0
intron: [130820321-130820447], 0
intron: [130820321-130820447], 0
intron: [130820321-130820447], 0
intron: [130820321-130820447], 0
intron: [130820321-130820447], 0
intron: [130820321-130820447], 0
intron: [130820321-130820447], 0
intron: [130820321-130820447], 0
intron: [130820321-130820447], 0
intron: [130820321-130820447], 0
chr9:130812841-130820923 Creating compatibility graph
chr9:130812841-130820923 Performing final collapse round
Extracted 8 contiguous transfrags from 8 scaffolds
chr9:130812841-130820923 Starting new collapse round
Convergence reached in 14 iterations
Importance sampling posterior distribution

ERROR: Multi-Hit not found (123409527,123409562).


Brute - force deleting the entry should probably help, but I would like to know why the program crashes for not havin found this multihit, instead of just ignoring it..

Thanks
Shrutii

[jgarbe]

I get the same Multi-Hit error as well while analyzing Illumina paired end data.
I'm using cufflinks 1.1.0 linked against Boost version 104700 on bam files generated by tophat 1.3.3.
I'm running command line: cufflinks -p 4 -u -b Sscrofa10.2.fa accepted_hits.bam

Half of my 10 samples run just fine, 4 fail with the Multi-hit error, and one generates a segmentation fault, all during the MLE step. I have found that I only get these errors when using the -u/--multi-read-correct option (with or without the -b option).

[HilmarBerger]

This line in the output

Number of Multi-Reads: 1 (with 458350 total hits)

indicates that there is only one multi-mapping read with a huge number of different mappings.
This could be caused by using the same query name for all reads (or a large part of it) in the input file. Please note that the SAM specification does assume that the string specified in the query name field identifies a read, the only exception being "*" if the query name is unknown.

Please check the input file and make sure that the query name (first column in SAM output) is either "*" or really something that identifies a read.

[glados]

I am bumping this few months old thread because I have a similar problem and in need of some help. I also get the "Multi-Hit not found"-error message on one of my test samples when using Cufflinks. I am using the --GTF-guide, --frag-bias-correct, --multi-read-correct and the --upper-quartile-norm parameters.

Here is the prompt output message:

You are using Cufflinks v1.3.0, which is the most recent release.
[13:19:01] Loading reference annotation.
[13:19:02] Inspecting reads and determining fragment length distribution.
> Processed 66947 loci.                        [*************************] 100%
> Map Properties:
>	Upper Quartile: 21.00
>	Number of Multi-Reads: 386605 (with 950358 total hits)
>	Fragment Length Distribution: Empirical (learned)
>	              Estimated Mean: 161.13
>	           Estimated Std Dev: 42.61
[13:21:07] Assembling transcripts and initializing abundances for multi-read correction.
> Processed 66947 loci.                        [*************************] 100%
[13:35:17] Loading reference annotation and sequence.
[13:35:27] Learning bias parameters.
> Processing Locus scaffold_4:12410956-1241332 [***********              ]  44%
WARNING: Multi-Hit not found (12414511,12416689).

WARNING: Multi-Hit not found (12414511,12416689).

WARNING: Multi-Hit not found (12414511,12416689).

WARNING: Multi-Hit not found (12414511,12416689).
> Processed 32442 loci.                        [*************************] 100%
[13:37:20] Re-estimating abundances with bias and multi-read correction.
> Processing Locus scaffold_4:12410956-1241332 [***********              ]  44%
WARNING: Multi-Hit not found (12414511,12416689).

WARNING: Multi-Hit not found (12414511,12416689).

WARNING: Multi-Hit not found (12414511,12416689).

WARNING: Multi-Hit not found (12414511,12416689).
> Processing Locus scaffold_4:12413851-1241839 [***********              ]  44%
WARNING: Multi-Hit not found (12414511,12416689).

WARNING: Multi-Hit not found (12414511,12416689).
> Processed 32442 loci.                        [*************************] 100%

When looking in the gtf-file I can not find a gene spanning those two exact coordinates. Question: Are these warning messages anything I should worry about? And why does it only occur for some samples?

[amitm]

I am bumping this few months old thread because I have a similar problem and in need of some help. I also get the "Multi-Hit not found"-error message on one of my test samples when using Cufflinks. I am using the --GTF-guide, --frag-bias-correct, --multi-read-correct and the --upper-quartile-norm parameters.


When looking in the gtf-file I can not find a gene spanning those two exact coordinates. Question: Are these warning messages anything I should worry about? And why does it only occur for some samples?

hi glados,
I am using Cufflinks 2.0.2 with GTF guide, frag-bias-correct, multi-read-correct, total-hits-norm on a Strand-specific human RNA-Seq data.
I am getting this same warning of multi-hit not found. Did you find answer to your query? As in yours, Cufflinks doesn't terminate, just returns warnings.
Should these be worried about? I read somewhere that this warning/ error is generated when using multi-read-correct. Is that right?

You are using Cufflinks v2.0.2, which is the most recent release.
[11:44:02] Loading reference annotation.
[11:44:06] Inspecting reads and determining fragment length distribution.
> Processed 155919 loci.                       [*************************] 100%
> Map Properties:
>	Normalized Map Mass: 57703206.56
>	Raw Map Mass: 57703206.56
>	Number of Multi-Reads: 1548338 (with 3999071 total hits)
>	Fragment Length Distribution: Empirical (learned)
>	              Estimated Mean: 157.64
>	           Estimated Std Dev: 32.05
[12:03:00] Assembling transcripts and initializing abundances for multi-read correction.
> Processed 155919 loci.                       [*************************] 100%
[14:23:07] Loading reference annotation and sequence.
[14:23:45] Learning bias parameters.
> Processing Locus chr16:222845-223709         [********                 ]  32%
WARNING: Multi-Hit not found (226782,227067).

WARNING: Multi-Hit not found (226782,227067).

WARNING: Multi-Hit not found (226782,227067).

WARNING: Multi-Hit not found (226782,227067).
> Processing Locus chr16:226678-227520         [********                 ]  32%
WARNING: Multi-Hit not found (226782,227067).

WARNING: Multi-Hit not found (226782,227067).
> Processing Locus chr8:6844699-6847243        [*********************    ]  85%
WARNING: Multi-Hit not found (6854483,6855187).

WARNING: Multi-Hit not found (6854483,6855187).

WARNING: Multi-Hit not found (6854483,6855187).

WARNING: Multi-Hit not found (6854483,6855187).
> Processing Locus chr8:6854287-6856724        [*********************    ]  85%
WARNING: Multi-Hit not found (6854483,6855187).

WARNING: Multi-Hit not found (6854483,6855187).
> Processed 26008 loci.                        [*************************] 100%
[14:41:54] Re-estimating abundances with bias and multi-read correction.
> Processing Locus chr16:222845-223709         [********                 ]  32%
WARNING: Multi-Hit not found (226782,227067).

WARNING: Multi-Hit not found (226782,227067).

WARNING: Multi-Hit not found (226782,227067).

WARNING: Multi-Hit not found (226782,227067).
> Processing Locus chr16:576846-577407         [********                 ]  32%
WARNING: Multi-Hit not found (226782,227067).

WARNING: Multi-Hit not found (226782,227067).
> Processing Locus chr8:6844699-6847243        [*********************    ]  85%
WARNING: Multi-Hit not found (6854483,6855187).

WARNING: Multi-Hit not found (6854483,6855187).

WARNING: Multi-Hit not found (6854483,6855187).

WARNING: Multi-Hit not found (6854483,6855187).
> Processing Locus chr8:6863802-6866346        [*********************    ]  85%
WARNING: Multi-Hit not found (6854483,6855187).

WARNING: Multi-Hit not found (6854483,6855187).
> Processed 26008 loci.                        [*************************] 100%

[glados]

Hello. I did not get any answers to that question, but I did not receive this warning in Cufflinks 2, so for me it's not a problem anymore at least. Hope you figure it out!