Simulated reads seem to not be compatible with Cell Ranger 6

[hoyu310]

Hi,
I tried running Cell Ranger 6 (latest version) on the simulated reads, but within two minutes before the alignment even began the following error message returned (the same run using Cell Ranger 3 completed successfully):

[error] Pipestance failed. Error log at:
simulate_rl151/SC_RNA_COUNTER_CS/SC_MULTI_CORE/MULTI_GEM_WELL_PROCESSOR/COUNT_GEM_WELL_PROCESSOR/_BASIC_SC_RNA_COUNTER/_MATRIX_COMPUTER/MAKE_SHARD/fork0/chnk0-u31d28af566/_errors

Log message:
stage failed unexpectedly: 'Invalid quality value 45 ASCII character 78 at position 0' execroot/home/git/checkouts/fastq_set-9504d5db1bfdb3fb/7cf23e7/src/squality.rs:19:
0: martian::martian_entry_point::{{closure}}
at execroot/exec/lib/rust/execroot/home/git/checkouts/martian-rust-35615836cc90309f/8b43658/martian/src/lib.rs:184:25
1: std::panicking::rust_panic_with_hook
at /rustc/e1884a8e3c3e813aada8254edfa120e85bf5ffca/library/std/src/panicking.rs:597:17
2: std::panicking::begin_panic_handler::{{closure}}
at /rustc/e1884a8e3c3e813aada8254edfa120e85bf5ffca/library/std/src/panicking.rs:499:13
3: std::sys_common::backtrace::__rust_end_short_backtrace
at /rustc/e1884a8e3c3e813aada8254edfa120e85bf5ffca/library/std/src/sys_common/backtrace.rs:141:18
4: rust_begin_unwind
at /rustc/e1884a8e3c3e813aada8254edfa120e85bf5ffca/library/std/src/panicking.rs:495:5
5: std::panicking::begin_panic_fmt
at /rustc/e1884a8e3c3e813aada8254edfa120e85bf5ffca/library/std/src/panicking.rs:437:5
6: <fastq_set::squality::SQualityContents as fastq_set::array::ArrayContent>::validate_bytes
at execroot/exec/lib/rust/execroot/home/git/checkouts/fastq_set-9504d5db1bfdb3fb/7cf23e7/execroot/home/git/checkouts/fastq_set-9504d5db1bfdb3fb/7cf23e7/src/squality.rs:19:17
fastq_set::array::ByteArray<N,T>::from_iter
at execroot/exec/lib/rust/execroot/home/git/checkouts/fastq_set-9504d5db1bfdb3fb/7cf23e7/src/array.rs:71:9
fastq_set::array::ByteArray<N,T>::new
at execroot/exec/lib/rust/execroot/home/git/checkouts/fastq_set-9504d5db1bfdb3fb/7cf23e7/src/array.rs:44:9
<cr_types::rna_read::RnaRead as cr_types::barcode::HasBarcode>::raw_bc_construct_qual::{{closure}}
at execroot/exec/lib/rust/cr_types/src/rna_read.rs:440:22
cr_types::barcode::BarcodeConstruct::map::{{closure}}
at execroot/exec/lib/rust/cr_types/src/barcode.rs:336:32
7: cr_types::barcode::BarcodeConstruct::map_result
at execroot/exec/lib/rust/cr_types/src/barcode.rs:320:43
cr_types::barcode::BarcodeConstruct::map
at execroot/exec/lib/rust/cr_types/src/barcode.rs:336:9
<cr_types::rna_read::RnaRead as cr_types::barcode::HasBarcode>::raw_bc_construct_qual
at execroot/exec/lib/rust/cr_types/src/rna_read.rs:439:9
8: <cr_lib::make_shard_metrics::MakeShardVisitor as cr_lib::barcode_sort::ReadVisitor>::visit_processed_read
at execroot/exec/lib/rust/cr_lib/src/make_shard_metrics.rs:261:47
cr_lib::barcode_sort::BarcodeSortWorkflow<Processor,SortOrder>::execute_workflow_with_visitor
at execroot/exec/lib/rust/cr_lib/src/barcode_sort.rs:106:21
<cr_lib::make_shard::MakeShard as martian::stage::MartianStage>::main
at execroot/exec/lib/rust/cr_lib/src/make_shard.rs:241:9
9: ::main
at execroot/exec/lib/rust/execroot/home/git/checkouts/martian-rust-35615836cc90309f/8b43658/martian/src/stage.rs:642:20
10: martian::martian_entry_point
at execroot/exec/lib/rust/execroot/home/git/checkouts/martian-rust-35615836cc90309f/8b43658/martian/src/lib.rs:225:9
martian::MartianAdapter::run_get_error
at execroot/exec/lib/rust/execroot/home/git/checkouts/martian-rust-35615836cc90309f/8b43658/martian/src/lib.rs:131:9
martian::MartianAdapter::run
at execroot/exec/lib/rust/execroot/home/git/checkouts/martian-rust-35615836cc90309f/8b43658/martian/src/lib.rs:124:9
cr_lib::main
at execroot/exec/lib/rust/cr_lib/src/bin/cr_lib.rs:111:23
11: core::ops::function::FnOnce::call_once
at /rustc/e1884a8e3c3e813aada8254edfa120e85bf5ffca/library/core/src/ops/function.rs:227:5
std::sys_common::backtrace::__rust_begin_short_backtrace
at /rustc/e1884a8e3c3e813aada8254edfa120e85bf5ffca/library/std/src/sys_common/backtrace.rs:125:18
12: main
13: __libc_start_main
14:
at /mnt/home/adam.azarchs/crosstool/build/src/glibc-2.12.2/csu/../sysdeps/x86_64/elf/start.S:103

[hiraksarkar]

Sorry for getting back to you late, but I think we don't support cellranger 6, it has to do with CB length in the fastq format. That could be an issue.

[hoyu310]

No problem, thanks for the reply.

[ColeWunderlich]

I think this is related to the sequence quality values that minnow is generating (using all Ns which corresponds to 78 in ascii)

Here's an example from a simulated R1.fastq

@AAAGAACAGACATGCGACGATGCTTTAA:GeneName:148:0:0
AAAGAACAGACATGCGACGATGCTTTAA
+
NNNNNNNNNNNNNNNNNNNNNNNNNNNN
@AAAGAACAGACATGCGGGTCCCGGGATG:GeneName:5:1:1
AAAGAACAGACATGCGGGTCCCGGGATG
+
NNNNNNNNNNNNNNNNNNNNNNNNNNNN

This corresponds to a quality value of 45 which is out of range for current illumina sequencers (illumina 1.8+, which tops out at J=41). (see here: https://en.m.wikipedia.org/wiki/FASTQ_format?useskin=vector#Encoding)

You can see that CR is complaining about quality scores from your error:

stage failed unexpectedly: 'Invalid quality value 45 ASCII character 78 at position 0

I think the solution would be to set them to something within the range of illumina 1.8 (say I or maybe H to be safe).