Alevin: Updates on How to Run 10x for v1 chemistry.

[kh49]

Alevin in Salmon v0.11.2

Describe the bug When attempting to run Alevin on https://support.10xgenomics.com/single-cell-gene-expression/datasets/1.1.0/cd14_monocytes, with the 10x v1 wrapper, the initialization seems to go smoothly then Alevin produces the following:

[2018-09-13 11:41:47.586] [jointLog] [info] Computed 0 rich equivalence classes for further processing
[2018-09-13 11:41:47.586] [jointLog] [info] Counted 0 total reads in the equivalence classes 
[2018-09-13 11:41:47.593] [jointLog] [warning] Only 0 fragments were mapped, but the number of burn-in fragments was set to 5000000.
The effective lengths have been computed using the observed mappings.

[2018-09-13 11:41:47.593] [jointLog] [warning] Something seems to be wrong with the calculation of the mapping rate.  The recorded ratio is likely wrong.  Please file this as a bug report.

[2018-09-13 11:41:47.593] [jointLog] [info] Mapping rate = 0%

To Reproduce Steps and data to reproduce the behavior:

Specifically, please provide at least the following information: A downloaded binary Salmon v0.11.2 was executed using the v1 wrapper script compiled locally in a Salmon specific Conda environment. The GRCh38.p12 reference was used. Dataset is linked above in bug description.

Full command used: ~/bin/salmon/scripts/v1_10x/run.sh salmon alevin -l ISR -1 read-I1_*.fastq.gz -2 read-RA_*.fastq.gz -r read-I1_*.fastq.gz --gemcode -i ../../../index_15_pc -p 10 -o ../../alevin_15_pc --tgMap ../../../txp2gene.tsv --dumpCsvCounts --dumpFeatures --end 5 --umiLength 5 --barcodeLength 14 Full terminal output:

(Salmonenv) @compute-1-16: ~/Documents/PBMCStem/cd14/fastqs/flowcell1 $ ~/bin/salmon/scripts/v1_10x/run.sh salmon alevin -l ISR -1 read-I1_*.fastq.gz -2 read-RA_*.fastq.gz -r read-I1_*.fastq.gz --gemcode  -i ../../../index_15_pc -p 10 -o ../../alevin_15_pc --tgMap ../../../txp2gene.tsv --dumpCsvCounts --dumpFeatures --end 5 --umiLength 5 --barcodeLength 14
TEMPDIR is /tmp/tmp.fyLlOm2tjU
Running command [salmon alevin -l ISR -1 read-I1_si-ACTTCACT_lane-001-chunk-001.fastq.gz read-I1_si-ACTTCACT_lane-002-chunk-000.fastq.gz read-I1_si-ACTTCACT_lane-003-chunk-003.fastq.gz read-I1_si-ACTTCACT_lane-004-chunk-002.fastq.gz read-I1_si-CGAAGTTG_lane-001-chunk-001.fastq.gz read-I1_si-CGAAGTTG_lane-002-chunk-000.fastq.gz read-I1_si-CGAAGTTG_lane-003-chunk-003.fastq.gz read-I1_si-CGAAGTTG_lane-004-chunk-002.fastq.gz read-I1_si-GAGCACGC_lane-001-chunk-001.fastq.gz read-I1_si-GAGCACGC_lane-002-chunk-000.fastq.gz read-I1_si-GAGCACGC_lane-003-chunk-003.fastq.gz read-I1_si-GAGCACGC_lane-004-chunk-002.fastq.gz read-I1_si-TTCGTGAA_lane-001-chunk-001.fastq.gz read-I1_si-TTCGTGAA_lane-002-chunk-000.fastq.gz read-I1_si-TTCGTGAA_lane-003-chunk-003.fastq.gz read-I1_si-TTCGTGAA_lane-004-chunk-002.fastq.gz -2 read-RA_si-ACTTCACT_lane-001-chunk-001.fastq.gz read-RA_si-ACTTCACT_lane-002-chunk-000.fastq.gz read-RA_si-ACTTCACT_lane-003-chunk-003.fastq.gz read-RA_si-ACTTCACT_lane-004-chunk-002.fastq.gz read-RA_si-CGAAGTTG_lane-001-chunk-001.fastq.gz read-RA_si-CGAAGTTG_lane-002-chunk-000.fastq.gz read-RA_si-CGAAGTTG_lane-003-chunk-003.fastq.gz read-RA_si-CGAAGTTG_lane-004-chunk-002.fastq.gz read-RA_si-GAGCACGC_lane-001-chunk-001.fastq.gz read-RA_si-GAGCACGC_lane-002-chunk-000.fastq.gz read-RA_si-GAGCACGC_lane-003-chunk-003.fastq.gz read-RA_si-GAGCACGC_lane-004-chunk-002.fastq.gz read-RA_si-TTCGTGAA_lane-001-chunk-001.fastq.gz read-RA_si-TTCGTGAA_lane-002-chunk-000.fastq.gz read-RA_si-TTCGTGAA_lane-003-chunk-003.fastq.gz read-RA_si-TTCGTGAA_lane-004-chunk-002.fastq.gz -r read-I1_si-ACTTCACT_lane-001-chunk-001.fastq.gz read-I1_si-ACTTCACT_lane-002-chunk-000.fastq.gz read-I1_si-ACTTCACT_lane-003-chunk-003.fastq.gz read-I1_si-ACTTCACT_lane-004-chunk-002.fastq.gz read-I1_si-CGAAGTTG_lane-001-chunk-001.fastq.gz read-I1_si-CGAAGTTG_lane-002-chunk-000.fastq.gz read-I1_si-CGAAGTTG_lane-003-chunk-003.fastq.gz read-I1_si-CGAAGTTG_lane-004-chunk-002.fastq.gz read-I1_si-GAGCACGC_lane-001-chunk-001.fastq.gz read-I1_si-GAGCACGC_lane-002-chunk-000.fastq.gz read-I1_si-GAGCACGC_lane-003-chunk-003.fastq.gz read-I1_si-GAGCACGC_lane-004-chunk-002.fastq.gz read-I1_si-TTCGTGAA_lane-001-chunk-001.fastq.gz read-I1_si-TTCGTGAA_lane-002-chunk-000.fastq.gz read-I1_si-TTCGTGAA_lane-003-chunk-003.fastq.gz read-I1_si-TTCGTGAA_lane-004-chunk-002.fastq.gz --gemcode -i ../../../index_15_pc -p 10 -o ../../alevin_15_pc --tgMap ../../../txp2gene.tsv --dumpCsvCounts --dumpFeatures --end 5 --umiLength 5 --barcodeLength 14 -1 /tmp/tmp.fyLlOm2tjU/p1.fa -2 /tmp/tmp.fyLlOm2tjU/p2.fa -r read-I1_si-ACTTCACT_lane-001-chunk-001.fastq.gz
read-I1_si-ACTTCACT_lane-002-chunk-000.fastq.gz
read-I1_si-ACTTCACT_lane-003-chunk-003.fastq.gz
read-I1_si-ACTTCACT_lane-004-chunk-002.fastq.gz
read-I1_si-CGAAGTTG_lane-001-chunk-001.fastq.gz
read-I1_si-CGAAGTTG_lane-002-chunk-000.fastq.gz
read-I1_si-CGAAGTTG_lane-003-chunk-003.fastq.gz
read-I1_si-CGAAGTTG_lane-004-chunk-002.fastq.gz
read-I1_si-GAGCACGC_lane-001-chunk-001.fastq.gz
read-I1_si-GAGCACGC_lane-002-chunk-000.fastq.gz
read-I1_si-GAGCACGC_lane-003-chunk-003.fastq.gz
read-I1_si-GAGCACGC_lane-004-chunk-002.fastq.gz
read-I1_si-TTCGTGAA_lane-001-chunk-001.fastq.gz
read-I1_si-TTCGTGAA_lane-002-chunk-000.fastq.gz
read-I1_si-TTCGTGAA_lane-003-chunk-003.fastq.gz
read-I1_si-TTCGTGAA_lane-004-chunk-002.fastq.gz]
Version Info: This is the most recent version of Salmon.
[2018-09-11 16:28:53.145] [alevinLog] [info] A custom protocol (END, BC length, UMI length) = (5, 14, 5) is being used.  Updating UMI k-mer length accordingly.
Logs will be written to ../../alevin_15_pc/logs
### salmon (single-cell-based) v0.11.2
### [ program ] => salmon 
### [ command ] => alevin 
### [ libType ] => { ISR }
### [ mates1 ] => { read-I1_si-ACTTCACT_lane-001-chunk-001.fastq.gz read-I1_si-ACTTCACT_lane-002-chunk-000.fastq.gz read-I1_si-ACTTCACT_lane-003-chunk-003.fastq.gz read-I1_si-ACTTCACT_lane-004-chunk-002.fastq.gz read-I1_si-CGAAGTTG_lane-001-chunk-001.fastq.gz read-I1_si-CGAAGTTG_lane-002-chunk-000.fastq.gz read-I1_si-CGAAGTTG_lane-003-chunk-003.fastq.gz read-I1_si-CGAAGTTG_lane-004-chunk-002.fastq.gz read-I1_si-GAGCACGC_lane-001-chunk-001.fastq.gz read-I1_si-GAGCACGC_lane-002-chunk-000.fastq.gz read-I1_si-GAGCACGC_lane-003-chunk-003.fastq.gz read-I1_si-GAGCACGC_lane-004-chunk-002.fastq.gz read-I1_si-TTCGTGAA_lane-001-chunk-001.fastq.gz read-I1_si-TTCGTGAA_lane-002-chunk-000.fastq.gz read-I1_si-TTCGTGAA_lane-003-chunk-003.fastq.gz read-I1_si-TTCGTGAA_lane-004-chunk-002.fastq.gz }
### [ mates2 ] => { read-RA_si-ACTTCACT_lane-001-chunk-001.fastq.gz read-RA_si-ACTTCACT_lane-002-chunk-000.fastq.gz read-RA_si-ACTTCACT_lane-003-chunk-003.fastq.gz read-RA_si-ACTTCACT_lane-004-chunk-002.fastq.gz read-RA_si-CGAAGTTG_lane-001-chunk-001.fastq.gz read-RA_si-CGAAGTTG_lane-002-chunk-000.fastq.gz read-RA_si-CGAAGTTG_lane-003-chunk-003.fastq.gz read-RA_si-CGAAGTTG_lane-004-chunk-002.fastq.gz read-RA_si-GAGCACGC_lane-001-chunk-001.fastq.gz read-RA_si-GAGCACGC_lane-002-chunk-000.fastq.gz read-RA_si-GAGCACGC_lane-003-chunk-003.fastq.gz read-RA_si-GAGCACGC_lane-004-chunk-002.fastq.gz read-RA_si-TTCGTGAA_lane-001-chunk-001.fastq.gz read-RA_si-TTCGTGAA_lane-002-chunk-000.fastq.gz read-RA_si-TTCGTGAA_lane-003-chunk-003.fastq.gz read-RA_si-TTCGTGAA_lane-004-chunk-002.fastq.gz }
### [ unmatedReads ] => { read-I1_si-ACTTCACT_lane-001-chunk-001.fastq.gz read-I1_si-ACTTCACT_lane-002-chunk-000.fastq.gz read-I1_si-ACTTCACT_lane-003-chunk-003.fastq.gz read-I1_si-ACTTCACT_lane-004-chunk-002.fastq.gz read-I1_si-CGAAGTTG_lane-001-chunk-001.fastq.gz read-I1_si-CGAAGTTG_lane-002-chunk-000.fastq.gz read-I1_si-CGAAGTTG_lane-003-chunk-003.fastq.gz read-I1_si-CGAAGTTG_lane-004-chunk-002.fastq.gz read-I1_si-GAGCACGC_lane-001-chunk-001.fastq.gz read-I1_si-GAGCACGC_lane-002-chunk-000.fastq.gz read-I1_si-GAGCACGC_lane-003-chunk-003.fastq.gz read-I1_si-GAGCACGC_lane-004-chunk-002.fastq.gz read-I1_si-TTCGTGAA_lane-001-chunk-001.fastq.gz read-I1_si-TTCGTGAA_lane-002-chunk-000.fastq.gz read-I1_si-TTCGTGAA_lane-003-chunk-003.fastq.gz read-I1_si-TTCGTGAA_lane-004-chunk-002.fastq.gz }
### [ gemcode ] => { }
### [ index ] => { ../../../index_15_pc }
### [ threads ] => { 10 }
### [ output ] => { ../../alevin_15_pc }
### [ tgMap ] => { ../../../txp2gene.tsv }
### [ dumpCsvCounts ] => { }
### [ dumpFeatures ] => { }
### [ end ] => { 5 }
### [ umiLength ] => { 5 }
### [ barcodeLength ] => { 14 }
### [ mates1 ] => { /tmp/tmp.fyLlOm2tjU/p1.fa }
### [ mates2 ] => { /tmp/tmp.fyLlOm2tjU/p2.fa }
### [ unmatedReads ] => { read-I1_si-ACTTCACT_lane-001-chunk-001.fastq.gz read-I1_si-ACTTCACT_lane-002-chunk-000.fastq.gz read-I1_si-ACTTCACT_lane-003-chunk-003.fastq.gz read-I1_si-ACTTCACT_lane-004-chunk-002.fastq.gz read-I1_si-CGAAGTTG_lane-001-chunk-001.fastq.gz read-I1_si-CGAAGTTG_lane-002-chunk-000.fastq.gz read-I1_si-CGAAGTTG_lane-003-chunk-003.fastq.gz read-I1_si-CGAAGTTG_lane-004-chunk-002.fastq.gz read-I1_si-GAGCACGC_lane-001-chunk-001.fastq.gz read-I1_si-GAGCACGC_lane-002-chunk-000.fastq.gz read-I1_si-GAGCACGC_lane-003-chunk-003.fastq.gz read-I1_si-GAGCACGC_lane-004-chunk-002.fastq.gz read-I1_si-TTCGTGAA_lane-001-chunk-001.fastq.gz read-I1_si-TTCGTGAA_lane-002-chunk-000.fastq.gz read-I1_si-TTCGTGAA_lane-003-chunk-003.fastq.gz read-I1_si-TTCGTGAA_lane-004-chunk-002.fastq.gz }

[2018-09-11 16:28:53.158] [jointLog] [info] Fragment incompatibility prior below threshold.  Incompatible fragments will be ignored.
[2018-09-11 16:28:53.158] [jointLog] [warning] You seem to have passed in both un-paired reads and paired-end reads. It is not currently possible to quantify hybrid library types in salmon.
[2018-09-11 16:28:53.164] [alevinLog] [info] Processing barcodes files (if Present) 

processed 535 Million barcodes

[2018-09-11 16:33:49.759] [alevinLog] [info] Done barcode density calculation.
[2018-09-11 16:33:49.759] [alevinLog] [info] # Barcodes Used: 534903498 / 535096394.
[2018-09-11 16:33:55.869] [alevinLog] [info] Knee found left boundary at  11740 
[2018-09-11 16:33:59.242] [alevinLog] [info] Gauss Corrected Boundary at  4345 
[2018-09-11 16:33:59.242] [alevinLog] [info] Learned InvCov: 713.683 normfactor: 1183.93
[2018-09-11 16:33:59.242] [alevinLog] [info] Total 5344(has 999 low confidence) barcodes
[2018-09-11 16:33:59.358] [alevinLog] [info] Done True Barcode Sampling
[2018-09-11 16:33:59.891] [alevinLog] [info] Done populating Z matrix
[2018-09-11 16:33:59.972] [alevinLog] [info] Done indexing Barcodes
[2018-09-11 16:33:59.972] [alevinLog] [info] Total Unique barcodes found: 4180559
[2018-09-11 16:33:59.972] [alevinLog] [info] Used Barcodes except Whitelist: 173007
[2018-09-11 16:34:00.783] [alevinLog] [info] Done with Barcode Processing; Moving to Quantify

[2018-09-11 16:34:00.784] [alevinLog] [info] parsing read library format
[2018-09-11 16:34:00.784] [jointLog] [info] There are 2 libraries.
[2018-09-11 16:34:00.868] [jointLog] [info] Loading Quasi index
[2018-09-11 16:34:00.876] [jointLog] [info] Loading 32-bit quasi index
[2018-09-11 16:34:00.876] [stderrLog] [info] Loading Suffix Array 
[2018-09-11 16:34:18.777] [stderrLog] [info] Loading Transcript Info 
[2018-09-11 16:34:27.531] [stderrLog] [info] Loading Rank-Select Bit Array
[2018-09-11 16:34:27.735] [stderrLog] [info] There were 97465 set bits in the bit array
[2018-09-11 16:34:27.776] [stderrLog] [info] Computing transcript lengths
[2018-09-11 16:34:27.776] [stderrLog] [info] Waiting to finish loading hash
[2018-09-11 16:34:29.276] [stderrLog] [info] Done loading index
[2018-09-11 16:34:29.276] [jointLog] [info] done
[2018-09-11 16:34:29.276] [jointLog] [info] Index contained 97465 targets

[2018-09-11 16:34:30.109] [jointLog] [info] Computed 0 rich equivalence classes for further processing
[2018-09-11 16:34:30.109] [jointLog] [info] Counted 0 total reads in the equivalence classes 
[2018-09-11 16:34:30.116] [jointLog] [warning] Only 0 fragments were mapped, but the number of burn-in fragments was set to 5000000.
The effective lengths have been computed using the observed mappings.

[2018-09-11 16:34:30.116] [jointLog] [warning] Something seems to be wrong with the calculation of the mapping rate.  The recorded ratio is likely wrong.  Please file this as a bug report.

[2018-09-11 16:34:30.116] [jointLog] [info] Mapping rate = 0%

[2018-09-11 16:34:30.116] [jointLog] [info] finished quantifyLibrary()
[2018-09-11 16:34:30.156] [alevinLog] [info] Starting optimizer

Desktop (please complete the following information):

• OS: CentOS
• x86_64 x86_64 x86_64 GNU/Linux LSB Version: :base-4.0-amd64:base-4.0-noarch:core-4.0-amd64:core-4.0-noarch:graphics-4.0-amd64:graphics-4.0-noarch:printing-4.0-amd64:printing-4.0-noarch Distributor ID: CentOS Description: CentOS release 6.9 (Final) Release: 6.9 Codename: Final

[k3yavi]

Hi @pophipi , Sorry for the late response. We have updated some of the steps for the v1 pipeline and the relevant How to document has been updated too. Please refer to the tutorial and let us know if you still face the problem.

Specifically in your case you might have to do the following things:

• compile the wrapper.cpp file in salmon/scripts/v1_10x folder with the command:

  g++ -std=c++11 -O3 -I \<PATH TO SALMON INCLUDE DIRECTORY\>-o wrapper wrapper.cpp -lz

• Update the run.sh file here with the path to the wrapper binary created in the above step.
• Use following command to run alevin in v1 mode:

  ./run.sh salmon alevin -l ISR -b ./reads/ --gemcode -i ./index_15_pc -p 10 -o ./alevin_15_pc --tgMap ./txp2gene.tsv --dumpCsvCounts --dumpFeatures --end 5 --umiLength 5 --barcodeLength 14

[kh49]

@k3yavi That seems to have fixed it, but I did get a warning at the end:

[2018-09-18 15:18:46.675] [alevinLog] [info] Finished optimizer
[2018-09-18 15:18:46.697] [jointLog] [warning] NOTE: Read Lib [./read-I1_si-AGGGACTG_lane-001-chunk-001.fastq.gz, ./read-I1_si-AGGGACTG_lane-002-chunk-000.fastq.gz, ./read-I1_si-AGGGACTG_lane-003-chunk-003.fastq.gz, ./read-I1_si-AGGGACTG_lane-004-chunk-002.fastq.gz, ./read-I1_si-CCTCTAAC_lane-001-chunk-001.fastq.gz, ./read-I1_si-CCTCTAAC_lane-002-chunk-000.fastq.gz, ./read-I1_si-CCTCTAAC_lane-003-chunk-003.fastq.gz, ./read-I1_si-CCTCTAAC_lane-004-chunk-002.fastq.gz, ./read-I1_si-GACAGGCT_lane-001-chunk-001.fastq.gz, ./read-I1_si-GACAGGCT_lane-002-chunk-000.fastq.gz, ./read-I1_si-GACAGGCT_lane-003-chunk-003.fastq.gz, ./read-I1_si-GACAGGCT_lane-004-chunk-002.fastq.gz, ./read-I1_si-TTATCTGA_lane-001-chunk-001.fastq.gz, ./read-I1_si-TTATCTGA_lane-002-chunk-000.fastq.gz, ./read-I1_si-TTATCTGA_lane-003-chunk-003.fastq.gz, ./read-I1_si-TTATCTGA_lane-004-chunk-002.fastq.gz] :

Greater than 5% of the fragments disagreed with the provided library type; check the file: ../../alevin_15_pc/lib_format_counts.json for details

Is this ok to ignore?

[k3yavi]

can you share the contents of the file ../../alevin_15_pc/lib_format_counts.json ? Basically what it's saying is that the assumption made to explicitly define the library type in the command line flag i.e. -lISR which means that the library is stranded and the reads are coming from the reverse strand is getting violated. In 10x protocols we generally expects that the read follow the ISR standard but it looks like some 5% of the reads are not following this property and that's what Alevin is complaining. It is possible since this is v1, we might expect some non-trivial fraction of reads to be non-stranded but too hard to say .

[kh49]

It looks like the combined read files all got classified under "SF". I inspected the fastqs and they seem to be formatted as expected: read-RA files contain the 98bp transcript and 5bp umi reads, read-I1 contains the 14bp I7 barcode.

{
    "read_files": "./read-I1_si-ACTTCACT_lane-001-chunk-001.fastq.gz, ./read-I1_si-ACTTCACT_lane-002-chunk-000.fastq.gz, ./read-I1_si-ACTTCACT_lane-003-chunk-003.fastq.gz, ./read-I1_si-ACTTCACT_lane-004-chunk-002.fastq.gz, ./read-I1_si-CGAAGTTG_lane-001-chunk-001.fastq.gz, ./read-I1_si-CGAAGTTG_lane-002-chunk-000.fastq.gz, ./read-I1_si-CGAAGTTG_lane-003-chunk-003.fastq.gz, ./read-I1_si-CGAAGTTG_lane-004-chunk-002.fastq.gz, ./read-I1_si-GAGCACGC_lane-001-chunk-001.fastq.gz, ./read-I1_si-GAGCACGC_lane-002-chunk-000.fastq.gz, ./read-I1_si-GAGCACGC_lane-003-chunk-003.fastq.gz, ./read-I1_si-GAGCACGC_lane-004-chunk-002.fastq.gz, ./read-I1_si-TTCGTGAA_lane-001-chunk-001.fastq.gz, ./read-I1_si-TTCGTGAA_lane-002-chunk-000.fastq.gz, ./read-I1_si-TTCGTGAA_lane-003-chunk-003.fastq.gz, ./read-I1_si-TTCGTGAA_lane-004-chunk-002.fastq.gz",
    "expected_format": "U",
    "compatible_fragment_ratio": 0.0,
    "num_compatible_fragments": 0,
    "num_assigned_fragments": 162343601,
    "num_frags_with_consistent_mappings": 0,
    "num_frags_with_inconsistent_or_orphan_mappings": 0,
    "strand_mapping_bias": NaN,
    "MSF": 0,
    "OSF": 0,
    "ISF": 0,
    "MSR": 0,
    "OSR": 0,
    "ISR": 0,
    "SF": 0,
    "SR": 0,
    "MU": 0,
    "OU": 0,
    "IU": 0,
    "U": 0,
    "read_files": "( /tmp/tmp.EWJ7aRZf0W/p1.fa, /tmp/tmp.EWJ7aRZf0W/p2.fa )",
    "expected_format": "ISR",
    "compatible_fragment_ratio": 1.0,
    "num_compatible_fragments": 162343601,
    "num_assigned_fragments": 162343601,
    "num_frags_with_consistent_mappings": 0,
    "num_frags_with_inconsistent_or_orphan_mappings": 592460922,
    "MSF": 0,
    "OSF": 0,
    "ISF": 0,
    "MSR": 0,
    "OSR": 0,
    "ISR": 0,
    "SF": 592460922,
    "SR": 0,
    "MU": 0,
    "OU": 0,
    "IU": 0,
    "U": 0
}

read-RA sample:

@NB500915:115:HVHT5BGXX:1:11101:22700:1088 1:N:0:0
CTTCATGCCCTGGGTTCTGCCCGCACGGACCCCCATCTCTGTGACTTCCTGGAGACTCACTTCCTAGATGAGGAAGTGAAGCTTATCAAGAAGATGGG
+
A/AAAEEEEEE/EEE/EEEEE/EEAAE/EEE<EAAEE/EAEAEEEAE6E//EE///<6E//<EAEA/EEEEEEEAEA</EAA6EEEEEEE<EE/<//6
@NB500915:115:HVHT5BGXX:1:11101:22700:1088 4:N:0:0
CTGCG
+
AA/A6
@NB500915:115:HVHT5BGXX:1:11101:24667:1088 1:N:0:0
ATTGTCCGCCTGGATTCCCAGAAGCACATCGACTTCTCTCTGCGCTCTCCCTACGGGGGTGGCCGCCCGGGCCGCGTGAAGAGGAAGAATGCCAAGAA

read-I1 sample:

@NB500915:115:HVHT5BGXX:1:11101:22700:1088 2:N:0:0
TTATTCCTTGCCTC
+
A/AAAAEAAEEAEE
@NB500915:115:HVHT5BGXX:1:11101:24667:1088 2:N:0:0
GCTCACTGTAGTCG
+
AAAAAEEEEEEEEE
@NB500915:115:HVHT5BGXX:1:11101:10530:1088 2:N:0:0
TTGTCATGGGTGAG

[k3yavi]

Hi @pophipi , The numbers looks good, it was my mistake the libtypes in Alevin are tangled because of Single-end reads and naming clash with salmon. We will work on this sometime soon but as per the issue for running Alevin with v1 it looks good. I am closing this issue for now feel free to open it if you have any other questions .