found no concordant and consistent mapping using Salmon

Question

[keren.danan]

Hi,

I am new to bulkRNA-seq and trying to convert my fastq files to count matrix so I could run a differential analysis. I have read it is suggested to use Salmon. I have a few questions:

1. My data consists of healthy and control patients, so I created an index to salmon using the gencode human transcriptome and mentioned in the command to create the index --gencode:

   salmon index -t gencode.v41.transcripts.fa.gz -i salmon_index --gencode

When running quant method:

salmon quant -i salmon_index --libType A -1 ${prefix}_R1_001.fastq.gz -2 ${prefix}_R2_001.fastq.gz -o /bulkRNAseq_human/INCPMPM-14146.0/quant_samples/quant/${prefix};

I got a warning for all of my samples saying : "found no concordant and consistent mappings". When looking at some of the metadata info, I found most of them with above 90% mapped. Is it a problem? should I change anything?

1. If I want to use salmon on bulk RNA data from mice but I see there is only one read for example, one fastq file look like this : FGC2321_s_1_AGCTCGCT-GCAGAATC.fastq, how am I supposed to run the quant method?

Thanks :)

[ATpoint]

What is the read length of the experiment? Please show the salmon logs. Single-end quantification for the 2nd question is covered in the salmon manual.

[keren.danan]

after running : gunzip -c H1_S11_L001_R1_001.fastq.gz | awk 'NR%4 == 2 {lengths[length($0)]++} END {for (l in lengths) {print l, lengths[l]}}' It is printed - 67 9145551. About the logs:

[2022-07-17 14:34:08.236] [jointLog] [info] setting maxHashResizeThreads to 96
[2022-07-17 14:34:08.236] [jointLog] [info] Fragment incompatibility prior below threshold.  Incompatible fragments will be ignored.
[2022-07-17 14:34:08.236] [jointLog] [info] Usage of --validateMappings implies use of minScoreFraction. Since not explicitly specified, it is being set to 0.65
[2022-07-17 14:34:08.236] [jointLog] [info] Setting consensusSlack to selective-alignment default of 0.35.
[2022-07-17 14:34:08.236] [jointLog] [info] parsing read library format
[2022-07-17 14:34:08.236] [jointLog] [info] There is 1 library.
[2022-07-17 14:34:08.236] [jointLog] [info] Loading pufferfish index
[2022-07-17 14:34:08.236] [jointLog] [info] Loading dense pufferfish index.
[2022-07-17 14:34:09.413] [jointLog] [info] done
[2022-07-17 14:34:09.463] [jointLog] [info] Index contained 250,381 targets
[2022-07-17 14:34:09.507] [jointLog] [info] Number of decoys : 0
[2022-07-17 14:34:24.335] [fileLog] [info]
At end of round 0
==================
Observed 8335066 total fragments (8335066 in most recent round)

[2022-07-17 14:34:24.334] [jointLog] [info] Computed 426,076 rich equivalence classes for further processing
[2022-07-17 14:34:24.334] [jointLog] [info] Counted 7,629,092 total reads in the equivalence classes
[2022-07-17 14:34:24.485] [jointLog] [info] Number of mappings discarded because of alignment score : 1,020,939
[2022-07-17 14:34:24.485] [jointLog] [info] Number of fragments entirely discarded because of alignment score : 143,414
[2022-07-17 14:34:24.485] [jointLog] [info] Number of fragments discarded because they are best-mapped to decoys : 0
[2022-07-17 14:34:24.485] [jointLog] [info] Number of fragments discarded because they have only dovetail (discordant) mappings to valid targets : 0
[2022-07-17 14:34:24.485] [jointLog] [info] Mapping rate = 91.5301%

[2022-07-17 14:34:24.485] [jointLog] [info] finished quantifyLibrary()
[2022-07-17 14:34:24.489] [jointLog] [info] Starting optimizer
[2022-07-17 14:34:24.663] [jointLog] [info] Marked 0 weighted equivalence classes as degenerate
[2022-07-17 14:34:24.671] [jointLog] [info] iteration = 0 | max rel diff. = 2580.12
[2022-07-17 14:34:25.353] [jointLog] [info] iteration = 100 | max rel diff. = 18.1319
[2022-07-17 14:34:26.025] [jointLog] [info] iteration = 200 | max rel diff. = 19.3459
[2022-07-17 14:34:26.696] [jointLog] [info] iteration = 300 | max rel diff. = 15.3254
[2022-07-17 14:34:27.368] [jointLog] [info] iteration = 400 | max rel diff. = 12.866
[2022-07-17 14:34:28.040] [jointLog] [info] iteration = 500 | max rel diff. = 0.283219
[2022-07-17 14:34:28.712] [jointLog] [info] iteration = 600 | max rel diff. = 5.67953
[2022-07-17 14:34:29.383] [jointLog] [info] iteration = 700 | max rel diff. = 0.208217
[2022-07-17 14:34:30.053] [jointLog] [info] iteration = 800 | max rel diff. = 4.05825
[2022-07-17 14:34:30.725] [jointLog] [info] iteration = 900 | max rel diff. = 2.69145
[2022-07-17 14:34:31.396] [jointLog] [info] iteration = 1,000 | max rel diff. = 0.333874
[2022-07-17 14:34:32.066] [jointLog] [info] iteration = 1,100 | max rel diff. = 0.0407324
[2022-07-17 14:34:32.147] [jointLog] [info] iteration = 1,113 | max rel diff. = 0.00810947
[2022-07-17 14:34:32.163] [jointLog] [info] Finished optimizer
[2022-07-17 14:34:32.163] [jointLog] [info] writing output

[2022-07-17 14:34:32.499] [jointLog] [warning] NOTE: Read Lib [[ H15_S1_L001_R1_001.fastq.gz, H15_S1_L001_R2_001.fastq.gz]] :

Found no concordant and consistent mappings. If this is a paired-end library, are you sure the reads are properly paired? 

[Rob]

Can you provide the contents of the file lib_format_counts.json?

[keren.danan]

"read_files": "[ H15N_S2_L001_R1_001.fastq.gz, H15N_S2_L001_R2_001.fastq.gz]",
"expected_format": "IU",
"compatible_fragment_ratio": 1.0,
"num_compatible_fragments": 7165735,
"num_assigned_fragments": 7165735,
"num_frags_with_concordant_consistent_mappings": 0,
"num_frags_with_inconsistent_or_orphan_mappings": 7440128,
"strand_mapping_bias": 0.0,
"MSF": 0,
"OSF": 0,
"ISF": 0,
"MSR": 0,
"OSR": 0,
"ISR": 0,
"SF": 3777656,
"SR": 3662472,
"MU": 0,
"OU": 0,
"IU": 0,
"U": 0

[Rob]

Thanks! So it looks like you have reads mapping but almost always as orphans. You may want to look at running your data through repair.sh from the BBmap toolkit to "repair" or re-synchronize the files, as it looks like they may may somehow have gotten desynchronized.

[ATpoint]

That having said, dod you manipulate the data somehow, e.g. trimming, renaming of reads, something like that?

[keren.danan]

No, this is the raw data I received. I will try to use repair.sh than. Thanks!