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Long DNA reads aligned
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10 months ago
tarek.mohamed • 250

Hi

What are the best long reads aligners? I have dna sequencing data generated by minion. Which aligners should I use, I need to detect SNPs and indels downstream

Thanks

nanopore • 475 views
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9 months ago
Belgium

Minimap2 is the currently recommended aligner for long read DNA and cDNA/RNA sequencing. If you are specifically interested in large structural variation you should also try ngmlr.

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I used Minimap2 to align my minion reads to a reference genome. I then tried to sort it using picard but I got an error. Why there is no sequence for this read? $ java -jar /Users/tarekmagdyshehatamohamed/miniconda3/envs/bioinfo/share/picard-2.17.0-0/picard.jar SortSam I=BC01_minimap.sam O=BC01_aln_sorted.bam SORT_ORDER=coordinate

Exception in thread "main" htsjdk.samtools.SAMFormatException: Error parsing text SAM file. CIGAR covers 442 bases but the sequence is 0 read bases ; File BC01_minimap.sam; Line 518
Line: 726b2822-4c7f-4812-8e6f-e21f4629d92b  272 chr17   1661134 0   6S4M1D75M1D15M1I29M2D12M1I8M1D5M2I13M1I11M2I71M1D11M1D81M1D29M2D28M1I13M1I8M1I2M3D1M1D10M   *   0   0   *   *   NM:i:33 ms:i:678    AS:i:678    nn:i:0  tp:A:S  cm:i:31 s1:i:265    dv:f:0.0533

Here is the complete error message

20:45:39.629 INFO  NativeLibraryLoader - Loading libgkl_compression.dylib from jar:file:/Users/tarekmagdyshehatamohamed/miniconda3/envs/bioinfo/share/picard-2.17.0-0/picard.jar!/com/intel/gkl/native/libgkl_compression.dylib
[Tue Jul 17 20:45:39 CDT 2018] SortSam INPUT=BC01_minimap.sam OUTPUT=BC01_aln_sorted.bam SORT_ORDER=coordinate    VERBOSITY=INFO QUIET=false VALIDATION_STRINGENCY=STRICT COMPRESSION_LEVEL=5 MAX_RECORDS_IN_RAM=500000 CREATE_INDEX=false CREATE_MD5_FILE=false GA4GH_CLIENT_SECRETS=client_secrets.json USE_JDK_DEFLATER=false USE_JDK_INFLATER=false
[Tue Jul 17 20:45:39 CDT 2018] Executing as tarekmagdyshehatamohamed@FSMD25VN00GJ1GN on Mac OS X 10.13.5 x86_64; OpenJDK 64-Bit Server VM 1.8.0_121-b15; Deflater: Intel; Inflater: Intel; Picard version: 2.17.0-SNAPSHOT
[Tue Jul 17 20:45:39 CDT 2018] picard.sam.SortSam done. Elapsed time: 0.00 minutes.
Runtime.totalMemory()=257425408
To get help, see http://broadinstitute.github.io/picard/index.html#GettingHelp
Exception in thread "main" htsjdk.samtools.SAMFormatException: Error parsing text SAM file. CIGAR covers 442 bases but the sequence is 0 read bases ; File BC01_minimap.sam; Line 518
Line: 726b2822-4c7f-4812-8e6f-e21f4629d92b  272 chr17   1661134 0   6S4M1D75M1D15M1I29M2D12M1I8M1D5M2I13M1I11M2I71M1D11M1D81M1D29M2D28M1I13M1I8M1I2M3D1M1D10M   *   0   0   *   *   NM:i:33 ms:i:678    AS:i:678    nn:i:0  tp:A:S  cm:i:31 s1:i:265    dv:f:0.0533
    at htsjdk.samtools.SAMLineParser.reportErrorParsingLine(SAMLineParser.java:457)
    at htsjdk.samtools.SAMLineParser.parseLine(SAMLineParser.java:355)
    at htsjdk.samtools.SAMTextReader$RecordIterator.parseLine(SAMTextReader.java:268)
    at htsjdk.samtools.SAMTextReader$RecordIterator.next(SAMTextReader.java:255)
    at htsjdk.samtools.SAMTextReader$RecordIterator.next(SAMTextReader.java:228)
    at htsjdk.samtools.SamReader$AssertingIterator.next(SamReader.java:576)
    at htsjdk.samtools.SamReader$AssertingIterator.next(SamReader.java:548)
    at picard.sam.SortSam.doWork(SortSam.java:100)
    at picard.cmdline.CommandLineProgram.instanceMain(CommandLineProgram.java:268)
    at picard.cmdline.PicardCommandLine.instanceMain(PicardCommandLine.java:98)
    at picard.cmdline.PicardCommandLine.main(PicardCommandLine.java:108)
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That read (flag 272) is not a primary alignment, therefore the sequence is not reported (twice).

Just use samtools for sorting, and you can do it simultaneously with alignment and avoid intermediate files:

minimap2 -t 8 -a yourgenome.fa yourreads.fastq.gz | samtools sort -@8 -o youralignment.bam

In my example I used 8 threads for alignment and sorting, which you'll have to adapt for your system.

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I am trying to call SNPs and indels, but all the tools I found are compatible with bam files generated with specific aligners. Is there a universal SNP caller that an work with minimap2. I do not want to use nanopolish since it works only with fast5 files. I am currently testing nanosv, is there any other suggestions?

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@WouterDeCoster Sorry if this sounds silly, second command "samtools sort -@8 -o youralignment.bam" doesn't seem to work. It instead says samtools sort [options] <in.bam> <out.prefix>, whereas I have input file in .sam format. I have also tried with "minimap2 -ax map-ont -L -t 8". Is there something I missed/misunderstood? Thanks!

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Make sure you have a reasonably recent version of samtools. It seems you have an older version, which does not support the syntax I described above.

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