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Errors in SAM/BAM files can be diagnosed with ValidateSamFile

edited December 2017 in Solutions to Problems

The problem

You're trying to run a GATK or Picard tool that operates on a SAM or BAM file, and getting some cryptic error that doesn't clearly tell you what's wrong. Bits of the stack trace (the pile of lines in the output log that the program outputs when there is a problem) may contain the following: java.lang.String, Error Type Count, NullPointerException -- or maybe something else that doesn't mean anything to you.

Why this happens

The most frequent cause of these unexplained problems is not a bug in the program -- it's an invalid or malformed SAM/BAM file. This means that there is something wrong either with the content of the file (something important is missing) or with its format (something is written the wrong way). Invalid SAM/BAM files generally have one or more errors in the following sections: the header tags, the alignment fields, or the optional alignment tags. In addition, the SAM/BAM index file can be a source of errors as well.

The source of these errors is usually introduced by upstream processing tools, such as the genome mapper/aligner or any other data processing tools you may have applied before feeding the data to Picard or GATK.

The solution

To fix these problems, you first have to know what's wrong. Fortunately there's a handy Picard tool that can test for (almost) all possible SAM/BAM format errors, called ValidateSamFile.

We recommend the workflow included below for diagnosing problems with ValidateSamFile. This workflow will help you tackle the problem efficiently and set priorities for dealing with multiple errors (which often happens). We also outline typical solutions for common errors, but note that this is not meant to be an exhaustive list -- there are too many possible problems to tackle all of them in this document. To be clear, here we focus on diagnostics, not treatment.

In some cases, it may not be possible to fix some problems that are too severe, and you may need to redo the genome alignment/mapping from scratch! Consider running ValidateSamFile proactively at all key steps of your analysis pipeline to catch errors early!

Workflow for diagnosing SAM/BAM file errors with ValidateSamFile


1. Generate summary of errors

First, run ValidateSamFile in SUMMARY mode in order to get a summary of everything that is missing or improperly formatted in your input file. We set MODE=SUMMARY explicitly because by default the tool would just emit details about the 100 first problems it finds then quit. If you have some minor formatting issues that don't really matter but affect every read record, you won't get to see more important problems that occur later in the file.

$ java -jar picard.jar ValidateSamFile \ 
        I=input.bam \ 

If this outputs No errors found, then your SAM/BAM file is completely valid. If you were running this purely as a preventative measure, then you're good to go and proceed to the next step in your pipeline. If you were doing this to diagnose a problem, then you're back to square one -- but at least now you know it's not likely to be a SAM/BAM file format issue. One exception: some analysis tools require Read Group tags like SM that not required by the format specification itself, so the input files will pass validation but the analysis tools will still error out. If that happens to you, check whether your files have SM tags in the @RG lines in their BAM header. That is the most common culprit.

However, if the command above outputs one or more of the 8 possible WARNING or 48 possible ERROR messages (see tables at the end of this document), you must proceed to the next step in the diagnostic workflow.

When run in SUMMARY mode, ValidateSamFile outputs a table that differentiates between two levels of error: ERROR proper and WARNING, based on the severity of problems that they would cause in downstream analysis. All problems that fall in the ERROR category must be addressed to in order to proceed with other Picard or GATK tools, while those that fall in the WARNING category may often be ignored for some, if not all subsequent analyses.

Example of error summary

ValidateSamFile (SUMMARY) Count

This table, generated by ValidateSamFile from a real BAM file, indicates that this file has a total of 1 MISSING_READ_GROUP error, 4 MISMATCH_MATE_ALIGNMENT_START errors, 894,289 MATES_ARE_SAME_END errors, and so on. Moreover, this output also indicates that there are 54 RECORD_MISSING_READ_GROUP warnings and 33 MISSING_TAG_NM warnings.

2. Generate detailed list of ERROR records

Since ERRORs are more severe than WARNINGs, we focus on diagnosing and fixing them first. From the first step we only had a summary of errors, so now we generate a more detailed report with this command:

$ java -jar picard.jar ValidateSamFile \ 
        I=input.bam \ 
        IGNORE_WARNINGS=true \

Note that we invoked the MODE=VERBOSE and the IGNORE_WARNINGS=true arguments.

The former is technically not necessary as VERBOSE is the tool's default mode, but we specify it here to make it clear that that's the behavior we want. This produces a complete list of every problematic record, as well as a more descriptive explanation for each type of ERROR than is given in the SUMMARY output.

The IGNORE_WARNINGS option enables us to specifically examine only the records with ERRORs. When working with large files, this feature can be quite helpful, because there may be many records with WARNINGs that are not immediately important, and we don't want them flooding the log output.

Example of VERBOSE report for ERRORs only

ValidateSamFile (VERBOSE) Error Description
ERROR: Read groups is empty Empty read group field for multiple records
ERROR: Record 1, Read name 20FUKAAXX100202:6:27:4968:125377Mate alignment does not match alignment start of mate
ERROR: Record 3, Read name 20FUKAAXX100202:6:27:4986:125375 Both mates are marked as second of pair
ERROR: Record 6, Read name 20GAVAAXX100126:4:47:18102:194445 Read CIGAR M operator maps off end of reference
ERROR: Read name 30PPJAAXX090125:1:60:1109:517#0 Mate not found for paired read
ERROR: Record 402, Read name 20GAVAAXX100126:3:44:17022:23968 Mate unmapped flag does not match read unmapped flag of mate
ERROR: Record 12, Read name HWI-ST1041:151:C7BJEACXX:1:1101:1128:82805 Read length does not match quals length

These ERRORs are all problems that we must address before using this BAM file as input for further analysis. Most ERRORs can typically be fixed using Picard tools to either correct the formatting or fill in missing information, although sometimes you may want to simply filter out malformed reads using Samtools.

For example, MISSING_READ_GROUP errors can be solved by adding the read group information to your data using the AddOrReplaceReadGroups tool. Most mate pair information errors can be fixed with FixMateInformation.

Once you have attempted to fix the errors in your file, you should put your new SAM/BAM file through the first validation step in the workflow, running ValidateSamFile in SUMMARY mode again. We do this to evaluate whether our attempted fix has solved the original ERRORs, and/or any of the original WARNINGs, and/or introduced any new ERRORs or WARNINGs (sadly, this does happen).

If you still have ERRORs, you'll have to loop through this part of the workflow until no more ERRORs are detected.

If you have no more ERRORs, congratulations! It's time to look at the WARNINGs (assuming there are still some -- if not, you're off to the races).

3. Generate detailed list of WARNING records

To obtain more detailed information about the warnings, we invoke the following command:

$ java -jar picard.jar ValidateSamFile \ 
        I=input.bam \ 
        IGNORE=type \

At this time we often use the IGNORE option to tell the program to ignore a specific type of WARNING that we consider less important, in order to focus on the rest. In some cases we may even decide to not try to address some WARNINGs at all because we know they are harmless (for example, MATE_NOT_FOUND warnings are expected when working with a small snippet of data). But in general we do strongly recommend that you address all of them to avoid any downstream complications, unless you're sure you know what you're doing.

Example of VERBOSE report for WARNINGs only

ValidateSamFile (VERBOSE) Warning Description
WARNING: Read name H0164ALXX140820:2:1204:13829:66057 A record is missing a read group
WARNING: Record 1, Read name HARMONIA-H16:1253:0:7:1208:15900:108776 NM tag (nucleotide differences) is missing

Here we see a read group-related WARNING which would probably be fixed when we fix the MISSING_READ_GROUP error we encountered earlier, hence the prioritization strategy of tackling ERRORs first and WARNINGs second.

We also see a WARNING about missing NM tags. This is an alignment tag that is added by some but not all genome aligners, and is not used by the downstream tools that we care about, so you may decide to ignore this warning by adding IGNORE=MISSING_TAG_NM from now on when you run ValidateSamFile on this file.

Once you have attempted to fix all the WARNINGs that you care about in your file, you put your new SAM/BAM file through the first validation step in the workflow again, running ValidateSamFile in SUMMARY mode. Again, we check that no new ERRORs have been introduced and that the only WARNINGs that remain are the ones we feel comfortable ignoring. If that's not the case we run through the workflow again. If it's all good, we can proceed with our analysis.

Appendix: List of all WARNINGs and ERRORs emitted by ValidateSamFile

The following two tables describe WARNING (Table I) and ERROR (Table II) cases, respectively.

Table I
Header Issues
INVALID_DATE_STRINGDate string is not ISO-8601
INVALID_QUALITY_FORMAT Quality encodings out of range; appear to be Solexa or Illumina when Phred expected. Avoid exception being thrown as a result of no qualities being read.
General Alignment Record Issues
ADJACENT_INDEL_IN_CIGAR CIGAR string contains an insertion (I) followed by deletion (D), or vice versa
RECORD_MISSING_READ_GROUP A SAMRecord is found with no read group id
Mate Pair Issues
PAIRED_READ_NOT_MARKED_AS_FIRST_OR_SECOND Pair flag set but not marked as first or second of pair
Optional Alignment Tag Issues
MISSING_TAG_NM The NM tag (nucleotide differences) is missing
E2_BASE_EQUALS_PRIMARY_BASE Secondary base calls should not be the same as primary, unless one or the other is N
General File, Index or Sequence Dictionary Issues
BAM_FILE_MISSING_TERMINATOR_BLOCK BAM appears to be healthy, but is an older file so doesn't have terminator block
Table II
ERROR Description
Header Issues
DUPLICATE_PROGRAM_GROUP_ID Same program group id appears more than once
DUPLICATE_READ_GROUP_ID Same read group id appears more than once
HEADER_RECORD_MISSING_REQUIRED_TAG Header tag missing in header line
HEADER_TAG_MULTIPLY_DEFINED Header tag appears more than once in header line with different value
INVALID_PLATFORM_VALUE The read group has an invalid value set for its PL field
INVALID_VERSION_NUMBER Does not match any of the acceptable versions
MISSING_HEADER The SAM/BAM file is missing the header
MISSING_PLATFORM_VALUE The read group is missing its PL (platform unit) field
MISSING_READ_GROUP The header is missing read group information
MISSING_SEQUENCE_DICTIONARY There is no sequence dictionary in the header
MISSING_VERSION_NUMBER Header has no version number
POORLY_FORMATTED_HEADER_TAG Header tag does not have colon
READ_GROUP_NOT_FOUND A read group ID on a SAMRecord is not found in the header
UNRECOGNIZED_HEADER_TYPE Header record is not one of the standard types
General Alignment Record Issues
CIGAR_MAPS_OFF_REFERENCE Bases corresponding to M operator in CIGAR extend beyond reference
INVALID_ALIGNMENT_START Alignment start position is incorrect
INVALID_CIGAR CIGAR string error for either read or mate
INVALID_FLAG_FIRST_OF_PAIR First of pair flag set for unpaired read
INVALID_FLAG_SECOND_OF_PAIR Second of pair flag set for unpaired read
INVALID_FLAG_PROPER_PAIR Proper pair flag set for unpaired read
INVALID_FLAG_MATE_NEG_STRAND Mate negative strand flag set for unpaired read
INVALID_FLAG_NOT_PRIM_ALIGNMENT Not primary alignment flag set for unmapped read
INVALID_FLAG_SUPPLEMENTARY_ALIGNMENT Supplementary alignment flag set for unmapped read
INVALID_FLAG_READ_UNMAPPED Mapped read flat not set for mapped read
INVALID_INSERT_SIZE Inferred insert size is out of range
INVALID_MAPPING_QUALITY Mapping quality set for unmapped read or is >= 256
MISMATCH_READ_LENGTH_AND_QUALS_LENGTH Length of sequence string and length of base quality string do not match
TAG_VALUE_TOO_LARGE Unsigned integer tag value is deprecated in BAM. Template length
Mate Pair Issues
INVALID_FLAG_MATE_UNMAPPED Mate unmapped flag is incorrectly set
MATE_NOT_FOUND Read is marked as paired, but its pair was not found
MATE_CIGAR_STRING_INVALID_PRESENCE A cigar string for a read whose mate is NOT mapped
MATE_FIELD_MISMATCH Read alignment fields do not match its mate
MATES_ARE_SAME_END Both mates of a pair are marked either as first or second mates
MISMATCH_FLAG_MATE_UNMAPPED Mate unmapped flag does not match read unmapped flag of mate
MISMATCH_FLAG_MATE_NEG_STRAND Mate negative strand flag does not match read strand flag
MISMATCH_MATE_ALIGNMENT_START Mate alignment does not match alignment start of mate
MISMATCH_MATE_CIGAR_STRING The mate cigar tag does not match its mate's cigar string
MISMATCH_MATE_REF_INDEX Mate reference index (MRNM) does not match reference index of mate
Optional Alignment Tag Issues
INVALID_MATE_REF_INDEX Mate reference index (MRNM) set for unpaired read
INVALID_TAG_NM The NM tag (nucleotide differences) is incorrect
MISMATCH_READ_LENGTH_AND_E2_LENGTH Lengths of secondary base calls tag values and read should match
MISMATCH_READ_LENGTH_AND_U2_LENGTH Secondary base quals tag values should match read length
EMPTY_READ Indicates that a read corresponding to the first strand has a length of zero and/or lacks flow signal intensities (FZ)
INVALID_INDEXING_BIN Indexing bin set on SAMRecord does not agree with computed value
General File, Index or Sequence Dictionary Issues
INVALID_INDEX_FILE_POINTER Invalid virtualFilePointer in index
INVALID_REFERENCE_INDEX Reference index not found in sequence dictionary
RECORD_OUT_OF_ORDER The record is out of order
TRUNCATED_FILE BAM file does not have terminator block
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