How do I load bam file and idx.fa file to the IGV browser?

Question

[melissachua90]

I want to load bam file and the human_refseq.idx.fa file to IGV. My questions are: (1) Should I be loading the indexed file? testes.index (2) Should the human_refseq.idx.fa file be loaded as the genome?

I will detail the steps that I used.

First, I obtained the primary assembly and created the RSEM reference:

rsem-refseq-extract-primary-assembly GCF_000001405.31_GRCh38.p5_genomic.fna GCF_000001405.31_GRCh38.p5_genomic.primary_assembly.fna

rsem-prepare-reference --gff3 GCF_000001405.31_GRCh38.p5_genomic.gff --bowtie2 --bowtie2-path ./bowtie2-2.4.5-py39hd2f7db1_2 --trusted-sources BestRefSeq GCF_000001405.31_GRCh38.p5_genomic.primary_assembly.fna human_refseq

I then calculated the expression and align the sequence:

rsem-calculate-expression -p 2 --output-genome-bam --bowtie2 --bowtie2-sensitivity-level "very_fast" --append-names --paired-end s_2_1_sequence.txt s_2_2_sequence.txt human_refseq testes

Next, I sorted and index the bam file:

samtools sort -l 9 -o testes.sorted.bam -@ 4 testes.transcript.bam
samtools index -bc -@ 4 testes.sorted.bam testes.index

Now, I want to use IGV. I want to load the bam file and human_refseq.idx.fa file to IGV. My attempt: (1) Go to the IGV. (2) Click "File"..."Load from file"...and then load the index file testes.index. (3) Click "Genomes"..."Load Genome from file"...upload the "human_refseq.idx.fa.fai" file.

I'm wondering if I did it correctly?

[Pierre Lindenbaum]

IGV wants a samtools fai index, this kind of index is unrelated to RSEM.

[melissachua90]

The testes.index is generated using samtools index command.

[Pierre Lindenbaum]

igv will look for a file named .fai, not .index

[melissachua90]

So I should load both the bam file and fai index file in addition to the human_refseq.idx.fa? Or just bam file and fai index (the latter as genome reference).

[GenoMax]

You do not need to provide testes.index file name to samtools index command. Process will automatically create a testes.sorted.bam.fai file using the name of BAM input.