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The GATK supports the BAM format for reads, quality scores, alignments, and metadata (e.g. the lane of sequencing, center of origin, sample name, etc.). No other file formats are supported.
The GATK doesn't have any tools for getting data into BAM format, but many other toolkits exist for this purpose. We recommend you look at Picard and Samtools for creating and manipulating BAM files. Also, many aligners are starting to emit BAM files directly. See BWA for one such aligner.
All BAM files must satisfy the following requirements:
See the BAM specification for more information.
It depends on whether you're using the NCBI/GRC build 36/build 37 version of the human genome, or the UCSC hg18/hg19 version of the human genome. While substantially equivalent, the naming conventions are different. The canonical ordering of contigs for these genomes is as follows:
Human genome reference consortium standard ordering and names (b3x):
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, X, Y, MT...
UCSC convention (hg1x):
chrM, chr1, chr2, chr3, chr4, chr5, chr6, chr7, chr8, chr9, chr10, chr11, chr12, chr13, chr14, chr15, chr16, chr17, chr18, chr19, chr20, chr21, chr22, chrX, chrY...
The easiest way to do it is to download Samtools and run the following command to examine the header of your file:
$ samtools view -H /path/to/my.bam @HD VN:1.0 GO:none SO:coordinate @SQ SN:1 LN:247249719 @SQ SN:2 LN:242951149 @SQ SN:3 LN:199501827 @SQ SN:4 LN:191273063 @SQ SN:5 LN:180857866 @SQ SN:6 LN:170899992 @SQ SN:7 LN:158821424 @SQ SN:8 LN:146274826 @SQ SN:9 LN:140273252 @SQ SN:10 LN:135374737 @SQ SN:11 LN:134452384 @SQ SN:12 LN:132349534 @SQ SN:13 LN:114142980 @SQ SN:14 LN:106368585 @SQ SN:15 LN:100338915 @SQ SN:16 LN:88827254 @SQ SN:17 LN:78774742 @SQ SN:18 LN:76117153 @SQ SN:19 LN:63811651 @SQ SN:20 LN:62435964 @SQ SN:21 LN:46944323 @SQ SN:22 LN:49691432 @SQ SN:X LN:154913754 @SQ SN:Y LN:57772954 @SQ SN:MT LN:16571 @SQ SN:NT_113887 LN:3994 ...
If the order of the contigs here matches the contig ordering specified above, and the
SO:coordinate flag appears in your header, then your contig and read ordering satisfies the GATK requirements.
Picard offers a tool called SortSam that will sort a BAM file properly. A similar utility exists in Samtools, but we recommend the Picard tool because SortSam will also set a flag in the header that specifies that the file is correctly sorted, and this flag is necessary for the GATK to know it is safe to process the data. Also, you can use the ReorderSam command to make a BAM file SQ order match another reference sequence.
A quick Unix command using Samtools will do the trick:
$ samtools view -H /path/to/my.bam | grep '^@RG' @RG ID:0 PL:solid PU:Solid0044_20080829_1_Pilot1_Ceph_12414_B_lib_1_2Kb_MP_Pilot1_Ceph_12414_B_lib_1_2Kb_MP LB:Lib1 PI:2750 DT:2008-08-28T20:00:00-0400 SM:NA12414 CN:bcm @RG ID:1 PL:solid PU:0083_BCM_20080719_1_Pilot1_Ceph_12414_B_lib_1_2Kb_MP_Pilot1_Ceph_12414_B_lib_1_2Kb_MP LB:Lib1 PI:2750 DT:2008-07-18T20:00:00-0400 SM:NA12414 CN:bcm @RG ID:2 PL:LS454 PU:R_2008_10_02_06_06_12_FLX01080312_retry LB:HL#01_NA11881 PI:0 SM:NA11881 CN:454MSC @RG ID:3 PL:LS454 PU:R_2008_10_02_06_07_08_rig19_retry LB:HL#01_NA11881 PI:0 SM:NA11881 CN:454MSC @RG ID:4 PL:LS454 PU:R_2008_10_02_17_50_32_FLX03080339_retry LB:HL#01_NA11881 PI:0 SM:NA11881 CN:454MSC ...
The presence of the
@RG tags indicate the presence of read groups. Each read group has a
SM tag, indicating the sample from which the reads belonging to that read group originate.
In addition to the presence of a read group in the header, each read must belong to one and only one read group. Given the following example reads,
$ samtools view /path/to/my.bam | grep '^@RG' EAS139_44:2:61:681:18781 35 1 1 0 51M = 9 59 TAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAA B<>;==?=?<==?=?=>>?>><=<?=?8<=?>?<:=?>?<==?=>:;<?:= RG:Z:4 MF:i:18 Aq:i:0 NM:i:0 UQ:i:0 H0:i:85 H1:i:31 EAS139_44:7:84:1300:7601 35 1 1 0 51M = 12 62 TAACCCTAAGCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAA G<>;==?=?&=>?=?<==?>?<>>?=?<==?>?<==?>?1==@>?;<=><; RG:Z:3 MF:i:18 Aq:i:0 NM:i:1 UQ:i:5 H0:i:0 H1:i:85 EAS139_44:8:59:118:13881 35 1 1 0 51M = 2 52 TAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAA @<>;<=?=?==>?>?<==?=><=>?-?;=>?:><==?7?;<>?5?<<=>:; RG:Z:1 MF:i:18 Aq:i:0 NM:i:0 UQ:i:0 H0:i:85 H1:i:31 EAS139_46:3:75:1326:2391 35 1 1 0 51M = 12 62 TAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAA @<>==>?>@???B>A>?>A?A>??A?@>?@A?@;??A>@7>?>>@:>=@;@ RG:Z:0 MF:i:18 Aq:i:0 NM:i:0 UQ:i:0 H0:i:85 H1:i:31 ...
membership in a read group is specified by the
RG:Z:* tag. For instance, the first read belongs to read group 4 (sample NA11881), while the last read shown here belongs to read group 0 (sample NA12414).
Yes! Many algorithms in the GATK need to know that certain reads were sequenced together on a specific lane, as they attempt to compensate for variability from one sequencing run to the next. Others need to know that the data represents not just one, but many samples. Without the read group and sample information, the GATK has no way of determining this critical information.
For technical details, see the SAM specification on the Samtools website.
|Tag||Importance||SAM spec definition||Meaning|
||Required||Read group identifier. Each
||Ideally, this should be a globally unique identify across all sequencing data in the world, such as the Illumina flowcell + lane name and number. Will be referenced by each read with the
||Sample. Use pool name where a pool is being sequenced.||Required. As important as
||The name of the sample sequenced in this read group. GATK tools treat all read groups with the same
||Platform/technology used to produce the read. Valid values: ILLUMINA, SOLID, LS454, HELICOS and PACBIO.||Important. Not currently used in the GATK, but was in the past, and may return. The only way to known the sequencing technology used to generate the sequencing data .||It's a good idea to use this field.|
||DNA preparation library identify||Essential for MarkDuplicates||MarkDuplicates uses the LB field to determine which read groups might contain molecular duplicates, in case the same DNA library was sequenced on multiple lanes.|
We do not require value for the
A concrete example may be instructive. Suppose I have a trio of samples: MOM, DAD, and KID. Each has two DNA libraries prepared, one with 400 bp inserts and another with 200 bp inserts. Each of these libraries is run on two lanes of an Illumina HiSeq, requiring 3 x 2 x 2 = 12 lanes of data. When the data come off the sequencer, I would create 12 bam files, with the following
@RG fields in the header:
Dad's data: @RG ID:FLOWCELL1.LANE1 PL:ILLUMINA LB:LIB-DAD-1 SM:DAD PI:200 @RG ID:FLOWCELL1.LANE2 PL:ILLUMINA LB:LIB-DAD-1 SM:DAD PI:200 @RG ID:FLOWCELL1.LANE3 PL:ILLUMINA LB:LIB-DAD-2 SM:DAD PI:400 @RG ID:FLOWCELL1.LANE4 PL:ILLUMINA LB:LIB-DAD-2 SM:DAD PI:400 Mom's data: @RG ID:FLOWCELL1.LANE5 PL:ILLUMINA LB:LIB-MOM-1 SM:MOM PI:200 @RG ID:FLOWCELL1.LANE6 PL:ILLUMINA LB:LIB-MOM-1 SM:MOM PI:200 @RG ID:FLOWCELL1.LANE7 PL:ILLUMINA LB:LIB-MOM-2 SM:MOM PI:400 @RG ID:FLOWCELL1.LANE8 PL:ILLUMINA LB:LIB-MOM-2 SM:MOM PI:400 Kid's data: @RG ID:FLOWCELL2.LANE1 PL:ILLUMINA LB:LIB-KID-1 SM:KID PI:200 @RG ID:FLOWCELL2.LANE2 PL:ILLUMINA LB:LIB-KID-1 SM:KID PI:200 @RG ID:FLOWCELL2.LANE3 PL:ILLUMINA LB:LIB-KID-2 SM:KID PI:400 @RG ID:FLOWCELL2.LANE4 PL:ILLUMINA LB:LIB-KID-2 SM:KID PI:400
Note the hierarchical relationship between read groups (unique for each lane) to libraries (sequenced on two lanes) and samples (across four lanes, two lanes for each library).
Use Picard's AddOrReplaceReadGroups tool to add read group information.
You can use the GATK to do the following:
GATK -I full.bam -T PrintReads -L chr1:10-20 -o subset.bam
and you'll get a BAM file containing only reads overlapping those points. This operation retains the complete BAM header from the full file (this was the reference aligned to, after all) so that the BAM remains easy to work with. We routinely use these features for testing and high-performance analysis with the GATK.
Geraldine Van der Auwera, PhD